Yeast strains producing mammalian-like complex n-glycans

ABSTRACT

Described herein are methods and genetically engineered fungal cells useful for producing target molecules containing mammalian-like complex N-glycans or containing intermediates in a mammalian glycosylation pathway.

CROSS-REFERENCE TO RELATED

This application is a divisional, and claims priority, of co-pendingU.S. application Ser. No. 14/641,002, filed Mar. 6, 2015, which is acontinuation, and claims priority of U.S. application Ser. No.13/510,527, filed Oct. 31, 2012, now abandoned, which is a U.S. NationalStage application, and claims priority of International Application No.PCT/IB2010/003154, filed Nov. 19, 2010, 2002, which claims priority ofU.S. Provisional Application Ser. No. 61/262,828, filed Nov. 19, 2009.The contents of all of the prior applications are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The invention relates to methods and materials for producingglycoproteins in fungal cells, and more particularly, to geneticallyengineering fungal cells to produce proteins containing mammalian-likecomplex N-glycans or proteins containing intermediates within amammalian glycosylation pathway.

BACKGROUND

High performance expression systems are required to produce mostbiopharmaceuticals (e.g., recombinant proteins) currently underdevelopment. The biological activity of many of these biopharmaceuticalsis dependent on their post-translational modification (e.g.,phosphorylation or glycosylation). A yeast-based expression systemcombines the ease of genetic manipulation and fermentation of amicrobial organism with the capability to secrete and to modifyproteins. However, recombinant glycoproteins produced in yeast cellsexhibit mainly heterogeneous high-mannose and hyper-mannose glycanstructures, which can be detrimental to protein function, downstreamprocessing, and subsequent therapeutic use, particularly whereglycosylation plays a biologically significant role.

SUMMARY

The methods and genetically engineered fungal cells described herein canbe used to produce target molecules (e.g., target proteins) that containmammalian-like N-glycans or contain intermediates within the mammalian(e.g., human) glycosylation pathway. Target molecules isolated from suchengineered cells can be used for biopharmaceutical applicationsincluding antibody production, cytokine production, and for treatment ofmetabolic disorders such as lysosomal storage disorders.

In one aspect, this document features a method of producing a fungalcell (e.g., Yarrowia lipolytica or Arxula adeninivorans) capable ofproducing proteins comprising GlcNAcMan₅GlcNAc₂ N-glycans. The methodincludes providing a fungal cell genetically engineered to produceproteins comprising Man₅GlcNAc₂ N-glycans; and introducing into the cella nucleic acid encoding a GlcNAc-transferase I, wherein the nucleic acidincludes a nucleotide targeting sequence to target the encodedGlcNAc-transferase I to an intracellular compartment (e.g., Golgiapparatus), wherein expression of the GlcNAc-transferase I in the fungalcell produces proteins including GlcNAcMan₅GlcNAc₂ N-glycans. The methodfurther can include introducing into the cell a nucleic acid encoding atarget protein, wherein the cell produces the target protein modified toinclude the GlcNAcMan₅GlcNAc₂ N-glycans. The target protein can bind toan Fc receptor. The target protein can be an antibody or fragmentthereof. The target protein can be a therapeutic glycoprotein. Thetarget protein can be Interferon-β, GM-CSF, Interferon γ, orerythropoietin.

The fungal cell genetically engineered to produce proteins containingMan₅GlcNAc₂ N-glycans can be deficient in OCH1 activity and include anucleic acid encoding an α-1,2-mannosidase, wherein the nucleic acidencoding the α-1,2-mannosidase includes a nucleotide sequence encoding atargeting sequence to target the encoded α-1,2-mannosidase to theendoplasmic reticulum. The targeting sequence can be an HDEL sequence.

The method further can include introducing into a cell a nucleic acidencoding a mannosidase II, wherein the nucleic acid encoding themannosidase II includes a nucleotide sequence encoding a targetingsequence to target the encoded mannosidase II to the Golgi apparatus,wherein expression of the mannosidase II in the fungal cell producesproteins containing GlcNAcMan₃GlcNAc₂ N-glycans.

The method further can include introducing into a cell a nucleic acidencoding a galactosyltransferase, wherein the nucleic acid encoding thegalactosyltransferase includes a nucleotide sequence encoding atargeting sequence to target the encoded galactosyltransferase to theGolgi apparatus, wherein expression of the galactosyltransferase in thefungal cell produces proteins containing GalGlcNAcMan₅GlcNAc₂ orGalGlcNAcMan₃GlcNAc₂ N-glycans. The galactosyltransferase can be afusion of a UDP-Glc-4-epimerase and the catalytic domain of aβ-1,4-galactosyltransferase I. Such a method further can includeintroducing into the cell a nucleic acid encoding a target protein,wherein the cell produces the target protein modified to containGalGlcNAcMan₅GlcNAc₂ or GalGlcNAcMan₃GlcNAc₂ N-glycans. The methods caninclude isolating the target protein modified to contain theGalGlcNAcMan₅GlcNAc₂ or GalGlcNAcMan₃GlcNAc₂ N-glycans.

In another aspect, this document features a method of producing a targetprotein containing GlcNAcMan₃GlcNAc₂ N-glycans. The method includesproviding a fungal cell (e.g., Yarrowia lipolytica or Arxulaadeninivorans) genetically engineered to include a nucleic acid encodinga GlcNAc-transferase I, an α-1,2-mannosidase, and a mannosidase II,wherein the nucleic acid includes a nucleotide sequence encoding atargeting sequence, or nucleotide sequences encoding targetingsequences, to target each encoded protein to an intracellularcompartment, wherein the fungal cell is deficient in OCH1 activity; andintroducing into the cell a nucleic acid encoding a target protein,wherein the cell produces the target protein containingGlcNAcMan₃GlcNAc₂ N-glycans. The nucleic acid encoding theα-1,2-mannosidase can include an endoplasmic reticulum targetingsequence to target the encoded α-1,2-mannosidase to the endoplasmicreticulum. For example, the targeting sequence can be an HDEL sequence.The nucleic acid encoding the GlcNAc-transferase I and the mannosidaseII can include a Golgi targeting sequence, or Golgi targeting sequences,to target the encoded GlcNAc-transferase I and mannosidase II to theGolgi apparatus. The target protein can bind to an Fc receptor. Thetarget protein can be an antibody or fragment thereof. The targetprotein can be a therapeutic glycoprotein. The target protein can beInterferon-β, GM-CSF, Interferon γ, or erythropoietin.

In some embodiments, the method further can include introducing into thecell a nucleic acid encoding a galactosyltransferase, wherein thenucleic acid encoding the galactosyltransferase includes a nucleotidesequence encoding a targeting sequence to target the encodedgalactosyltransferase to the Golgi apparatus, wherein expression of thegalactosyltransferase in the fungal cell produces the target proteinmodified to contain GalGlcNAcMan₃GlcNAc₂ N-glycans. The target proteinmodified to contain GalGlcNAcMan₃GlcNAc₂ N-glycans can be isolated fromthe fungal cell.

This document also features a method of making a fungal cell (e.g.,Yarrowia lipolytica or Arxula adeninivorans) capable of producingproteins containing GlcNAcMan₃GlcNAc₂ N-glycans. The method includesproviding a fungal cell genetically engineered to produce proteinscontaining Man₃GlcNAc₂ N-glycans; introducing into the cell a nucleicacid encoding a GlcNAc-transferase I, wherein the nucleic acid includesa nucleotide sequence encoding a targeting sequence to target theencoded GlcNAc-transferase I to an intracellular compartment (e.g.,Golgi apparatus), wherein expression of the GlcNAc-transferase I in thefungal cell produces proteins containing GlcNAcMan₃GlcNAc₂ N-glycans.The method further can include introducing into the cell a nucleic acidencoding a target protein, wherein the cell produces the target proteinmodified to contain GlcNAcMan₃GlcNAc₂ N-glycans. The target protein canbind to an Fc receptor. The target protein can be an antibody orfragment thereof. The target protein can be a therapeutic glycoprotein.The target protein can be Interferon-β, GM-CSF, Interferon γ, orerythropoietin.

The fungal cell genetically engineered to produce proteins containingMan₃GlcNAc₂ N-glycans can be deficient in ALG3 activity, and include anucleic acid encoding an α-1,2-mannosidase, wherein the nucleic acidincludes a nucleotide sequence encoding a targeting sequence to targetthe encoded α-1,2-mannosidase to the endoplasmic reticulum. Such afungal cell further can be deficient in OCH1 activity and/or furtherinclude a nucleic acid encoding α-1,3-glucosyltransferase (e.g., ALG6).

The method further can include introducing into the cell a nucleic acidencoding a GlcNAc-transferase II, wherein the nucleic acid encoding theGlcNAc-transferase II includes a nucleotide sequence encoding atargeting sequence to target the encoded GlcNAc-transferase II to anintracellular compartment, wherein expression of the GlcNAc-transferaseII in the fungal cell produces proteins containing GlcNAc₂Man₃GlcNAc₂N-glycans.

The method further can include introducing into the cell a nucleic acidencoding a galactosyltransferase, wherein the nucleic acid encoding thegalactosyltransferase includes a nucleotide sequence encoding atargeting sequence to target the encoded galactosyltransferase to theGolgi apparatus, wherein expression of the galactosyltransferase in thefungal cell produces proteins containing GalGlcNAcMan₃GlcNAc₂ orGal₂GlcNAc₂Man₃GlcNAc₂ N-glycans. The galactosyltransferase can be afusion of a UDP-Glc-4-epimerase and catalytic domain of aβ-1,4-galactosyltransferase I. The method further can includeintroducing into the cell a nucleic acid encoding a target protein,wherein the cell produces the target protein modified to containGalGlcNAcMan₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

The method further can include introducing into the cell a nucleic acidencoding the α and β subunits of a Glucosidase II, wherein expression ofthe α and β subunits of the Glucosidase II in the fungal cell producesproteins including GalGlcNAcMan₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂N-glycans.

This document also features a method of producing a target proteincontaining Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans. The method includesproviding a fungal cell genetically engineered to be deficient in ALG3activity and including a nucleic acid encoding a GlcNAc-transferase I, aGlcNAc-transferase II, and a galactosyltransferase, wherein the nucleicacid encoding the GlcNAc-transferase I, the GlcNAc-transferase II, andthe galactosyltransferase include a nucleotide sequence encoding atargeting sequence, or nucleotide sequences encoding targetingsequences, to target each encoded protein to an intracellularcompartment (e.g., the Golgi apparatus); and introducing into the cell anucleic acid encoding a target protein, wherein the cell produces thetarget protein containing Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans. The fungalcell can be further deficient in OCH1 activity and/or further include anucleic acid encoding an α-1,3-glucosyltransferase such as ALG6. Thefungal cell further can include a nucleic acid encoding the α and βsubunits of a Glucosidase II, wherein expression of the α and β subunitsof the Glucosidase II in the fungal cell produces the target proteincontaining Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

In another aspect, this document features an isolated fungal cellgenetically engineered to produce proteins containing GlcNAcMan₃GlcNAc₂N-glycans. The fungal cell can be deficient in OCH1 activity and includea nucleic acid encoding an α-1,2-mannosidase, a GlcNAc-transferase I,and a mannosidase II, wherein the nucleic acid encoding theα-1,2-mannosidase, the GlcNAc-transferase I, and the mannosidase IIincludes a nucleotide sequence encoding a targeting sequence, ornucleotide sequences encoding targeting sequences, to target eachencoded protein to an intracellular compartment, wherein expression ofthe α-1,2-mannosidase, the GlcNAc-transferase I, and the mannosidase IIin the fungal cell produces proteins containing GlcNAcMan₃GlcNAc₂N-glycans. The fungal cell further can include a nucleic acid encoding atarget protein, wherein the cell produces the target protein modified tocontain GlcNAcMan₃GlcNAc₂ N-glycans.

In some embodiments, such a fungal cell further includes a nucleic acidencoding a GlcNAc-transferase II, wherein the nucleic acid encoding theGlcNAc-transferase II includes a nucleotide sequence encoding atargeting sequence to target the encoded GlcNAc-transferase II to anintracellular compartment, wherein expression of the GlcNAc-transferaseII in the fungal cell produces proteins containing GlcNAc₂Man₃GlcNAc₂N-glycans.

In some embodiments, such a fungal cell further includes a nucleic acidencoding a galactosyltransferase, wherein the nucleic acid encoding thegalactosyltransferase includes a nucleotide sequence encoding atargeting sequence to target the encoded galactosyltransferase to theGolgi apparatus, wherein expression of the galactosyltransferase in thefungal cell produces proteins containing Gal₂GlcNAc₂Man₃GlcNAc₂N-glycans.

In yet another aspect, this document features an isolated fungal cellgenetically engineered to produce proteins containing GlcNAc₂Man₃GlcNAc₂N-glycans. The fungal cell is genetically engineered to be deficient inALG3 activity and includes a nucleic acid encoding a GlcNAc-transferaseI and a GlcNAc-transferase II, wherein the nucleic acid encoding theGlcNAc-transferase I and the GlcNAc-transferase II includes a nucleotidesequence encoding a targeting sequence, or nucleotide sequences encodingtargeting sequences, to target each encoded protein to an intracellularcompartment, wherein expression of the GlcNAc-transferase I, and theGlcNAc-transferase II in the fungal cell produces proteins containingGlcNAc₂Man₃GlcNAc₂ N-glycans. The genetically engineered fungal cellfurther can be deficient in OCH1 activity and/or further include anucleic acid encoding an α-1,3-glucosyltransferase. A geneticallyengineered fungal cell also can include a nucleic acid encoding a targetprotein, wherein the cell produces the target protein modified tocontain GlcNAc₂Man₃GlcNAc₂ N-glycans. A fungal cell further can includea nucleic acid encoding the α and β subunits of a Glucosidase II,wherein expression of the α and β subunits of the Glucosidase II in thefungal cell produces the protein containing GlcNAc₂Man₃GlcNAc₂N-glycans. The fungal cell further can include a nucleic acid encoding agalactosyltransferase, wherein the nucleic acid encoding thegalactosyltransferase includes a nucleotide sequence encoding atargeting sequence to target the encoded galactosyltransferase to theGolgi apparatus, wherein expression of the galactosyltransferase in thefungal cell produces proteins containing Gal₂GlcNAc₂Man₃GlcNAc₂N-glycans.

This document also features a substantially pure culture of Yarrowialipolytica cells, a substantial number of which are geneticallyengineered to produce glycoproteins containing Gal₂GlcNac₂Man₃GlcNAc₂N-glycans. The cells are genetically engineered to be deficient in ALG3activity and include a nucleic acid encoding a GlcNAc-transferase I, aGlcNAc-transferase II, and a galactosyltransferase, wherein the nucleicacid encoding the GlcNAc-transferase I, the GlcNAc-transferase II, andthe galactosyltransferase include a nucleotide sequence encoding atargeting sequence, or nucleotides sequences encoding targetingsequences, to target each encoded protein to an intracellularcompartment, wherein expression of the GlcNAc-transferase I, theGlcNAc-transferase II, and the galactosyltransferase in the cellproduces proteins containing Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans. Thegenetically engineered fungal cell further can be deficient in OCH1activity and/or further include a nucleic acid encoding anα-1,3-glucosyltransferase (e.g., ALG6). The cells further can include anucleic acid encoding the α and β subunits of a Glucosidase II, whereinexpression of the α and β subunits of the Glucosidase II in the fungalcell produces the target protein containing Gal₂GlcNAc₂Man₃GlcNAc₂N-glycans.

In another aspect, this document features a substantially pure cultureof Yarrowia lipolytica cells, a substantial number of which aregenetically engineered to produce glycoproteins containingGal₂GlcNAc₂Man₃GlcNAc₂ N-glycans, wherein the cells are geneticallyengineered to be deficient in OCH1 activity and include a nucleic acidencoding an α-1,2-mannosidase, a GlcNAc-transferase I, a mannosidase II,a GlcNAc-transferase II, and a galactosyltransferase, wherein thenucleic acid encoding the α-1,2-mannosidase, the GlcNAc-transferase I,the mannosidase II, the GlcNAc-transferase II, and thegalactosyltransferase includes a nucleotide sequence encoding atargeting sequence, or nucleotide sequences encoding targetingsequences, to target each encoded protein to an intracellularcompartment, wherein expression of the α-1,2-mannosidase,GlcNAc-transferase I, mannosidase II, GlcNAc-transferase II, andgalactosyltransferase in the cells produces proteins comprisingGal₂GlcNAc₂Man₃GlcNAc₂ N-glycans.

This document also features a composition that includes a glycoprotein,wherein at least 50% (e.g., at least 70% or at least 85% of theN-glycans on the glycoprotein are GlcNAc₂Man₃GlcNAc₂ N-glycans.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the exemplary methods andmaterials are described below. All publications, patent applications,patents, Genbank® Accession Nos, and other references mentioned hereinare incorporated by reference in their entirety. In case of conflict,the present application, including definitions, will control. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a representation of Man₅GLcNAc₂ and Man₃GlcNAc₂ structures.

FIG. 1B is a schematic diagram of plasmid pYlOCH1 PUT TOPO.

FIG. 2 is a series of electroferograms depicting N-glycan analysis ofsecreted proteins obtained from po1d lnuga Yarrowia lipolytica wild-typecells or Δoch1 po1d lnuga Yarrowia lipolytica cells. The main N-glycanupon OCH1 inactivation becomes Man₅GlcNAc₂. Analysis was performed usingDNA sequencer-assisted, fluorophore-assisted carbohydrateelectrophoresis (DSA-FACE). “M5,” “M6,” “M8,” and “M9,” refer to thenumber of mannose residues conjugated to the base N-acetylglucosaminestructure. The Y-axis represents the relative fluorescence units as anindication of the amount of each N-glycan structure. The X-axisrepresents the relative mobility of each N-glycan structure through acapillary. The top electroferogram is an analysis of dextran for use asa mobility standard.

FIG. 3 is a schematic diagram of plasmids pYLHUXdL2preManHDEL andpYLTUXdL2preManHDEL.

FIG. 4 is a series of electroferograms depicting the N-glycan profileafter introduction of a ManHDEL (=HDEL-tagged α-1,2-mannosidase)expression cassette (either under TEF1 or Hp4d promoter control) intostrain G014. “Rd” stands for “random integration” via the zeta sequencespresent on the vectors shown in FIG. 3. The major N-glycan uponmannosidase expression is Man₅GlcNAc₂. Curing of the URA3 marker fromone of these strains (G018, see Table 2) does not change the N-glycanprofile.

FIG. 5 is a schematic of the construction strategy for plasmids JME926pPTLeu2-ADE2ex-Hp4dManHDEL(Yl) and OXYP289pPTAxp1-LEU2ex-Hp4dManHDEL(Yl). See FIG. 23 for the construction ofvector pYLTmAXrGnTII.

FIG. 6 is a series of electroferograms depicting the N-glycan profileafter introduction of a ManHDEL (=HDEL-tagged α-1,2-mannosidase)expression cassette (under Hp4d promoter control) into strain G014 bytargeted integration (Tg) in either the LEU2 or the AXP1 locus.Man₅GlcNAc₂ becomes the main N-glycan.

FIG. 7 is a depiction of the amino acid sequence (SEQ ID NO:3) andYarrowia codon optimized nucleotide sequence (SEQ ID NO:4) of the fusionprotein between the 100 N-terminal amino acids of Kre2p and thecatalytic domain of human GlcNAc-transferase I. In bold: Kre2p part offusion protein; in normal font: GnT I part of fusion protein;underlined: start and stop codons.

FIG. 8 is a schematic diagram of the construction strategy for plasmidspYLTmAXhGnTI and pYLHp4mAXhGnTI.

FIG. 9 is a series of electroferograms depicting the N-glycan profileafter introduction of the GnT I activity into strain G036 bytransformation with a vector expressing GnT I. “Rd” stands for “randomintegration” via the zeta sequences present on the vectors shown in FIG.8. The major N-glycan upon expression of the GnT I activity isGlcNAcMan₅GlcNAc₂. In vitro treatment with α-1,2-mannosidase does notchange the profile significantly, indicating that only small amounts ofhigh-mannose N-glycans other than Man₅GlcNAc₂ are present. In vitrohexosaminidase treatment results in a shift from GlcNAcMan₅GlcNAc₂towards Man₅GlcNAc₂.

FIG. 10 is a depiction of the amino acid sequence (SEQ ID NO:7) andYarrowia codon optimized nucleotide sequence (SEQ ID NO:8) of the fusionprotein between the 36 N-terminal amino acids of Mnn2p and the catalyticdomain of Drosophila melanogaster mannosidase II. In bold: Mnn2p part offusion protein; in normal font: Man II part of fusion protein;underlined: start and stop codons.

FIG. 11 is a schematic depiction of the construction strategy forplasmids pYLTmAXDmManII and pYLTmAXDmManII (LEU2ex).

FIG. 12 is a series of electroferograms depicting the N-glycan profileafter introduction of the Man II activity into strain G040 bytransformation with a Man II-expressing vector. “Rd” stands for “randomintegration” via the zeta sequences present on the vectors shown in FIG.11. Upon expression of the Man II activity a new peak appears withhigher electrophoretic mobility, as well as a ‘shoulder’ peak running atalmost the same position as Man₅GlcNAc₂. In vitro hexosaminidasetreatment results in a shift forward for these peaks (next to theobserved shift from GlcNAcMan₅GlcNAc₂ towards Man₅GlcNAc₂), indicatingthe presence of terminal GlcNAc and thus identifying the peaks asGlcNAcMan₃GlcNAc₂ and GlcNAcMan₄GlcNAc₂. In vitro treatment withα-1,2-mannosidase does not change the profile significantly, indicatingthat only small amounts of high-mannose N-glycans other than Man₅GlcNAc₂are present.

FIG. 13 is the amino acid sequence (SEQ ID NO:9) and Yarrowia codonoptimized nucleotide sequence (SEQ ID NO: 10) of the fusion proteinbetween the 46 N-terminal amino acids of Mnn2p, the Schizosaccharomycespombe UDP-Glc-4-epimerase-like protein and the catalytic domain of humanβ-1,4-galactosyl transferase I. The Mnn2p part of the fusion protein isfrom 1-46, linker sequences are from 47-49 and 405-408, epimerasesequences of the fusion protein are from 50-404, and the Man II part ofthe fusion protein is from 409-763 of SEQ ID NO:9. The Mnn2p part isfrom nucleotides 1-138, linker sequences are from nucleotides 139-147and 1213-1224, epimerase sequences are from nucleotides 148-1212, andMan II part is from 1225-2289 of SEQ ID NO:10. Start and stop codons areunderlined.

FIG. 14 is a schematic depiction of the construction strategy forplasmids pYLTmAXSpGal10hGalTI and pYLTmAXSpGal10hGalTI (ADE2ex).

FIG. 15 is a series of electroferograms depicting the N-glycan profileafter introduction of the Gal10-GalTI activity into strain G040. Theresulting transformant G044 was cultivated in 2 different media. “Rd”stands for “random integration” via the zeta sequences present on thevectors shown in FIG. 14. Upon expression of the Gal10-GalTI activity anew peak appears running at a position between Man₇GlcNAc₂ andMan₅GlcNAc₂. In vitro galactosidase treatment results in a shift forwardfor this peak and an equal increase of GlcNAcMan₅GlcNAc₂ (the latterbeing confirmed as representing this N-glycan by the double treatmentwith galactosidase and hexosaminidase). This indicates the presence ofterminal galactose and thus identifying the new peak of the G044 profileas GalGlcNAcMan₅GlcNAc₂. In vitro treatment with α-1,2-mannosidaseindicates the presence of a large amount of high-mannose N-glycans(especially Man₅GlcNAc₂) that were not yet trimmed to Man₅GlcNAc₂.

FIG. 16 is a schematic depiction of plasmid pYLalg3PUT-ALG6.

FIG. 17 is a series of electroferograms depicting the N-glycan profileafter introduction of pYLalg3PUT-ALG6 into strain G036. Overexpressionof ALG6 results in a significant amount of glucosylated peaks(GlcMan_(5′)GlcNAc₂ and Glc₂Man_(5′)GlcNAc₂), indicating that theGlc₃Man_(5′)GlcNAc₂ structure that was transferred to the nascentprotein is not completely trimmed towards Man_(5′)GlcNAc₂ by glucosidaseII. Depending on the growth medium, the generated Man_(5′)GlcNAc₂ ispartially (still some Man_(5′)GlcNAc₂ and Man_(4′)GlcNAc₂) or almostcompletely trimmed towards Man₃GlcNAc₂ by the action of the ER-localizedHDEL-tagged T. reesei α-1,2-mannosidase. The Man_(5′)GlcNAc₂ andMan_(4′)GlcNAc₂ peaks are identified as such, by their sensitivitytowards α-1,2-mannosidase. Because of the capping glucoses,GlcMan_(5′)GlcNAc₂ and Glc₂Man_(5′)GlcNAc₂ are insensitive towards thistreatment. Jack Bean mannosidase is partially capable of removing thefree α-1,6-linked mannose while it also converts Man_(3-5′)GlcNAc₂ intoMan₁GlcNAc₂.

FIG. 18 is a schematic depiction of the construction strategy forplasmid pYLTmAXhGnTI (Hygr ex).

FIG. 19 is a series of electroferograms depicting the N-glycan profilesafter introduction of the GnT I activity into either the non-cured(G039) or cured (G045) version of the Δalg3-Hp4dALG6 strain bytransformation with a GnT I-expressing vector. The generation ofGlcNAcMan₃GlcNAc₂ was proven via a hexosaminidase digest. The new peakcompletely shifts back towards Man₃GlcNAc₂. In strain G048 conversiontowards GlcNAcMan₃GlcNAc₂ was not complete since some Man₃GlcNAc₂ couldstill be observed. This strain also has some remnant Man_(5′)GlcNAc₂ asshown by the α-1,2-mannosidase digest.

FIG. 20 is a schematic depiction of the construction strategy forplasmid JME925 pPTAde2-URA3 ex-Hp4dhGnTI.

FIG. 21 is a series of electroferograms depicting N-glycan profilesafter introduction of the GnT I activity into the cured version of theΔalg3-Hp4dALG6 strain (=G045); integration of an Hp4d-driven expressionconstruct into the ADE2 locus (Tg-ade2). In this cultivation the amountof glucosylated N-glycans was high and conversion of Man_(4′-5′)GlcNAc₂to Man₃GlcNAc₂ was not complete. A new peak running next toMan_(4′)GlcNAc₂ was observed in transformant G057 and could bedesignated as GlcNAcMan₃GlcNAc₂ based on the result if thehexosaminidase digest: the new peak completely shifts back towardsMan₃GlcNAc₂.

FIG. 22 is the amino acid sequence (SEQ ID NO:17) and Yarrowia codonoptimized nucleotide sequence (SEQ ID NO: 18) of the fusion proteinbetween the 36 N-terminal amino acids of Mnn2p and the catalytic domainof rat GlcNAc-transferase II. In bold: Mnn2p part of fusion protein; innormal font: GnT II part of fusion protein; underlined: start and stopcodons.

FIG. 23 is a schematic depiction of the construction strategy forplasmids pYLTmAXrGnTII and pYLTmAXrGnTII (ADE2 ex).

FIG. 24 is a series of electroferograms depicting N-glycan profilesafter introduction of the GnT II activity into a strain synthesizingGlcNAcMan₃GlcNAc₂. The resulting strains were either obtained via doubletransformation of G045 with the GnTI and GnT II expression constructs orvia transformation of G047 with the GnTII expression construct. In bothcases, the peak representing GlcNAcMan₃GlcNAc₂ almost completelydisappeared and a new peak, about one glucose unit larger, appeared.Hexosaminidase treatment indicates the presence of two terminal GlcNAcresidues onto the new N-glycan; the peak shifts about two glucose unitsto the left and thus represents GlcNAc₂Man₃GlcNAc₂. α-1,2-mannosidasetreatment does not result into major differences, indicating that thereare only limited amounts of Man_(4′-5′)GlcNAc₂ present.

FIG. 25 is a schematic diagram of plasmids pYLTUXdL2preAnGlcII□ andpYLeu2ExTEFpreLip2AnGlucIIβ for expression of the glucosidase IIactivity.

FIG. 26 is a schematic of the construction strategy for plasmids JME923pPTura3-LEU2ex-TefL2preAnGlcIIa+b[alt1].

FIG. 27 is a schematic of the construction strategy for plasmids JME923pPTura3-LEU2ex-Hp4dL2preAnGlcIIa+b[alt1] andZeta-LEU2ex-Hp4dL2preAnGlcIIa+b[alt].

FIG. 28 is a series of electroferograms depicting N-glycan profilesafter introduction of the glucosidase II activity into a strainsynthesizing GlcNAcMan₃GlcNAc₂. The resulting strains were eitherobtained via random (G060) or targeted (G061) integration of a dualexpression construct for the gls2α and gls2β subunit. In both cases, areduction of glucosylated peaks is observed. α-1,2-mannosidase treatmentindicates that not all of the generated Man_(5′)GlcNAc₂ was convertedtowards Man₃GlcNAc₂ by the heterologous HDEL-tagged α-1,2-mannosidase.Because of the capping glucoses, GlcMan_(5′)GlcNAc₂ andGlc₂Man_(5′)GlcNAc₂ are insensitive towards this treatment. Jack Beanmannosidase is partially capable of removing the free α-1,6-linkedmannose on both the remaining glucosylated N-glycans andGlcNAcMan₃GlcNAc₂. Furthermore, this treatment converts Man₃-5′GlcNAc₂into Man₁GlcNAc₂. “Rd” stands for “random integration” via the zetasequences present on the vectors shown in FIG. 27. “Tg-ade2” and“Tg-ura3” stands for targeted integration in the ADE2 resp. URA3 locus.

FIG. 29 is a series of electroferograms depicting the N-glycan profileof the secretome of strains G070 and G071, which were generated via theintroduction of GlcNAc-transferase II into strain G061. The N-glycanswere treated with either α-1,2-mannosidase (removing all terminalα-1,2-linked mannose residues) or hexosaminidase (which removes terminalβ-1,2-linked GlcNAc residues) to allow identification of the peaks inthe G070 and G071 native profiles. The glucose-containing N-glycans arenot sensitive to either of the two enzymes. The α-1,2-mannosidasetreatment results in the trimming of Man₅′GlcNAc₂ and Man₄GlcNAc₂towards Man₃GlcNAc₂. The hexosaminidase treatment removes theβ-1,2-linked terminal GlcNAc residues that have been added byGlcNAc-transferase I and II to generate Man₃GlcNAc₂.

FIG. 30A is the nucleotide sequence of the synthetic preproLip2-lightchain (LC) (SEQ ID NO:32).

FIG. 30B is the amino acid sequence of the synthetic preproLip2-LC (SEQID NO:33)

FIG. 31A is the nucleotide sequence of the synthetic preproLip2-heavychain (HC) (SEQ ID NO:34).

FIG. 31B is the amino acid sequence of the synthetic preproLip2-HC (SEQID NO:35).

FIG. 32 is a series of electroferograms depicting the N-glycan profileanalysis of SuperT/glycerol shake-flask cultivations of glyco-engineeredstrains G045, G057, G061 and G071 that were transformed withpYLHp4L2preproHerHC/LC (GUT2ex)-ori2. See Table 2 for a description ofstrains G045, G057, G061 and G071.

FIG. 33 is a graph of the results from a functional ELISA at differenttime-points in the G096 fed-batch fermentation.

FIGS. 34A-1, 34A-2, 34B-1, and 34B-2 are a series of electroferogramsdepicting the N-glycan profile analysis of the secretome at differenttime-points within the G096 fed-batch fermentation.

DETAILED DESCRIPTION

As described herein, in vivo synthesis of mammalian-like complexN-glycans on yeast-secreted glycoproteins can be based on either aMan₅GlcNAc₂ or Man₃GlcNAc₂ base structure (see FIG. 1A, “Man” refers tomannose, and “GlcNAc” refers to N-glucosamine). To produce theMan₅GlcNAc₂ base structure, yeast cells can be engineered such thatα-1,2-mannosidase activity is increased in an intracellular compartmentand Outer CHain elongation (OCH1) activity is decreased. To produce theMan₃GlcNAc₂ base structure, activity of Asparagine Linked Glycosylation3 (ALG3) and, in some embodiments, OCH1 is decreased, activity ofα-1,2-mannosidase and, in some embodiments, activity ofα-1,3-glucosyltransferase is increased. The N-glycan profile of proteinsproduced in such yeast cells can be altered by further engineering theyeast cells to contain one or more of the following activities: GlcNActransferase I (GnT I) activity, mannosidase II activity, GlcNActransferase II (GnT II) activity, glucosidase II activity, andgalactosyltransferase (Gal T) activity. For example, expressing GnT I ina yeast cell producing Man₅GlcNAc₂ or Man₃GlcNAc₂ N-glycans results inthe transfer of a GlcNAc moiety to the Man₅GlcNAc₂ or Man₃GlcNAc₂N-glycans such that GlcNAcMan₅GlcNAc₂ or GlcNAcMan₃GlcNAc₂ N-glycans,respectively, are produced. In cells producing GlcNAcMan₅GlcNAc₂N-glycans, expressing a mannosidase II results in two mannose residuesbeing removed from GlcNAcMan₅GlcNAc₂ N-glycans to produceGlcNAcMan₃GlcNAc₂ N-glycans. In cells producing GlcNAcMan₃GlcNAc₂N-glycans, expressing GnT II results in the transfer of another GlcNAcmoiety to GlcNAcMan₃GlcNAc₂ N-glycans to produce GlcNAc₂Man₃GlcNAc₂N-glycans. Expressing Gal T in cells producing GlcNAcMan₃GlcNAc₂ orGlcNAc₂Man₃GlcNAc₂ N-glycans results in the transfer of galactose to theGlcNAcMan₃GlcNAc₂ or GlcNAc₂Man₃GlcNAc₂ N-glycans to produceGalGlcNAcMan₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans. In someembodiments, glucosidase II (e.g., by expressing α and β subunits) canbe expressed to increase production of the Man₃GlcNAc₂ base structure.

Target Molecules

Target molecules, as used herein, refer to any molecules that undergoN-glycosylation in a genetically engineered cell (e.g., a fungal cellsuch as Yarrowia lipolytica, Arxula adeninivorans, or other relatedspecies dimorphic yeast cell; a plant cell, or an animal cell). In someembodiments, the target molecules are capable of being traffickedthrough one or more steps of the Yarrowia lipolytica or Arxulaadeninivorans (or other related species dimorphic yeast) secretorypathway, resulting in their N-glycosylation by the host cell machinery.The target molecules can be endogenous or exogenous.

Suitable target proteins include pathogen proteins (e.g., tetanustoxoid; diptheria toxoid; viral surface proteins (e.g., cytomegalovirus(CMV) glycoproteins B, H and gCIII; human immunodeficiency virus 1(HIV-1) envelope glycoproteins; Rous sarcoma virus (RSV) envelopeglycoproteins; herpes simplex virus (HSV) envelope glycoproteins;Epstein Barr virus (EBV) envelope glycoproteins; varicella-zoster virus(VZV) envelope glycoproteins; human papilloma virus (HPV) envelopeglycoproteins; Influenza virus glycoproteins; and Hepatitis familysurface antigens), lysosomal proteins (e.g., glucocerebrosidase,cerebrosidase, or galactocerebrosidase), insulin, glucagon, growthfactors, cytokines, chemokines, a protein binding to an Fc receptor,antibodies or fragments thereof, or fusions of any of the proteins toantibodies or fragments of antibodies (e.g., protein-Fc). Growth factorsinclude, e.g., vascular endothelial growth factor (VEGF), Insulin-likegrowth factor (IGF), bone morphogenic protein (BMP), Granulocyte-colonystimulating factor (G-CSF), Granulocyte-macrophage colony stimulatingfactor (GM-CSF), Nerve growth factor (NGF); a Neurotrophin,Platelet-derived growth factor (PDGF), Erythropoietin (EPO),Thrombopoietin (TPO), Myostatin (GDF-8), Growth Differentiation factor-9(GDF9), basic fibroblast growth factor (bFGF or FGF2), Epidermal growthfactor (EGF), Hepatocyte growth factor (HGF). Cytokines include, e.g.,interleukins (e.g., IL-1 to IL-33 such as IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, or IL-15) and interferons(e.g., interferon (3 or interferon γ). Chemokines include, e.g., 1-309,TCA-3, MCP-1, MIP-1α, MIP-1β, RANTES, C10, MRP-2, MARC, MCP-3, MCP-2,MRP-2, CCF18, MIP-1γ, Eotaxin, MCP-5, MCP-4, NCC-1, Ckβ10, HCC-1,Leukotactin-1, LEC, NCC-4, TARC, PARC, or Eotaxin-2. Also included aretumor glycoproteins (e.g., tumor-associated antigens), for example,carcinoembryonic antigen (CEA), human mucins, HER-2/neu, andprostate-specific antigen (PSA) [Henderson and Finn, Advances inImmunology, 62, pp. 217-56 (1996)]. In one embodiment, the targetprotein is an anti-HER2/neu antibody. In some embodiments, the targetprotein can be one associated with a lysosomal storage disorder, whichtarget proteins include, e.g., alpha-L-iduronidase,beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase,beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfataseA, alpha-N-acetylgalactosaminidase, aspartylglucosaminidase,iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase,beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase,alpha-neuraminidase, phosphotransferase, acid lipase, acid ceramidase,sphingomyelinase, thioesterase, cathepsin K, and lipoprotein lipase.

Target proteins also can be fusion proteins. Fusions proteins include,e.g., a fusion of (i) any protein described herein or fragment thereofwith (ii) an antibody or fragment thereof. As used herein, the term“antibody fragment” refers to (a) an antigen-binding fragment or (b) anFc part of the antibody that can interact with an Fc receptor. Anantigen binding fragment can be, for example, a Fab, F(ab′)₂, Fv, andsingle chain Fv (scFv) fragment. An scFv fragment is a singlepolypeptide chain that includes both the heavy and light chain variableregions of the antibody from which the scFv is derived. In addition,diabodies [Poljak (1994) Structure 2(12):1121-1123; Hudson et al. (1999)J. Immunol. Methods 23(1-2):177-189] and intrabodies [Huston et al.(2001) Hum. Antibodies 10(3-4):127-142; Wheeler et al. (2003) Mol. Ther.8(3):355-366; Stocks (2004) Drug Discov. Today 9(22): 960-966] can beused in the methods of the invention.

Target proteins can also be joined to one or more of a polymer, acarrier, an adjuvant, an immunotoxin, or a detectable (e.g.,fluorescent, luminescent, or radioactive) moiety. For example, a targetprotein can be joined to polyethyleneglycol, which can be used toincrease the molecular weight of small proteins and/or increasecirculation residence time.

In some embodiments, the target molecule can be, or contain, dolichol.

Genetically Engineered Cells

Genetically engineered cells described herein can be used to producetarget molecules that contain mammalian-like N-glycans or targetmolecules that contain intermediates within the mammalian glycosylationpathway. For example, as described herein, nucleic acids encoding one ormore enzymes can be introduced into a fungal cell such that the cellproduces the desired N-glycan (e.g., GlcNAcMan₅GlcNAc₂,GlcNAcMan₃GlcNAc₂, GlcNAc₂Man₃GlcNAc₂, GalGlcNAcMan₃GlcNAc₂ orGal₂GlcNAc₂Man₃GlcNAc₂ N-glycans). Thus, in any of the embodimentsdescribed herein, a fungal cell may contain a nucleic acid encoding oneenzyme, or a nucleic acid may encode multiple enzymes. Each such nucleicacid also can contain a targeting sequence as discussed below. Inaddition, a nucleic acid encoding a target molecule also can beintroduced into the fungal cell such that the target molecule isproduced and modified to contain the desired N-glycan (e.g.,GlcNAcMan₅GlcNAc₂, GlcNAcMan₃GlcNAc₂, GlcNAc₂Man₃GlcNAc₂,GalGlcNAcMan₃GlcNAc₂ or Gal₂GlcNAc₂Man₃GlcNAc₂ N-glycans).

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein, and refer to both RNA and DNA, including cDNA, genomic DNA,synthetic DNA, and DNA (or RNA) containing nucleic acid analogs. Nucleicacids can have any three-dimensional structure. A nucleic acid can bedouble-stranded or single-stranded (i.e., a sense strand or an antisensestrand). Non-limiting examples of nucleic acids include genes, genefragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers, as well as nucleic acid analogs. “Polypeptide” and “protein”are used interchangeably herein and mean any peptide-linked chain ofamino acids, regardless of length or post-translational modification.

An “isolated nucleic acid” refers to a nucleic acid that is separatedfrom other nucleic acid molecules that are present in anaturally-occurring genome, including nucleic acids that normally flankone or both sides of the nucleic acid in a naturally-occurring genome(e.g., a yeast genome). The term “isolated” as used herein with respectto nucleic acids also includes any non-naturally-occurring nucleic acidsequence, since such non-naturally-occurring sequences are not found innature and do not have immediately contiguous sequences in anaturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., any paramyxovirus,retrovirus, lentivirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include an engineered nucleic acid such as a DNAmolecule that is part of a hybrid or fusion nucleic acid. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not considered an isolated nucleicacid.

The term “exogenous” as used herein with reference to nucleic acid and aparticular host cell refers to any nucleic acid that does not occur in(and cannot be obtained from) that particular cell as found in nature.Thus, a non-naturally-occurring nucleic acid is considered to beexogenous to a host cell once introduced into the host cell. It isimportant to note that non-naturally-occurring nucleic acids can containnucleic acid subsequences or fragments of nucleic acid sequences thatare found in nature provided that the nucleic acid as a whole does notexist in nature. For example, a nucleic acid molecule containing agenomic DNA sequence within an expression vector isnon-naturally-occurring nucleic acid, and thus is exogenous to a hostcell once introduced into the host cell, since that nucleic acidmolecule as a whole (genomic DNA plus vector DNA) does not exist innature. Thus, any vector, autonomously replicating plasmid, or virus(e.g., retrovirus, adenovirus, or herpes virus) that as a whole does notexist in nature is considered to be non-naturally-occurring nucleicacid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular cell. For example,an entire chromosome isolated from a cell of yeast x is an exogenousnucleic acid with respect to a cell of yeast y once that chromosome isintroduced into a cell of yeast.

Cells suitable for genetic engineering include, e.g., fungal cells(e.g., Yarrowia lipolytica or any other related dimorphic yeast cellsdescribed herein), plant cells, or animal cells. The cells can beprimary cells, immortalized cells, or transformed cells. The cells canbe those in an animal, e.g., a non-human mammal. Such cells, prior tothe genetic engineering as specified herein, can be obtained from avariety of commercial sources and research resource facilities, such as,for example, the American Type Culture Collection (Rockville, Md.).

Genetic engineering of a cell can include genetic modifications such as:(i) deletion of an endogenous gene encoding a protein havingN-glycosylation activity; (ii) introduction of a recombinant nucleicacid encoding a mutant form of a protein (e.g., endogenous or exogenousprotein) having N-glycosylation activity (i.e., expressing a mutantprotein having an N-glycosylation activity); (iii) introduction orexpression of an RNA molecule that interferes with the functionalexpression of a protein having the N-glycosylation activity; (iv)introduction of a recombinant nucleic acid encoding a wild-type (e.g.,endogenous or exogenous) protein having N-glycosylation activity (i.e.,expressing a protein having an N-glycosylation activity); or (v)altering the promoter or enhancer elements of one or more endogenousgenes encoding proteins having N-glycosylation activity to thus alterthe expression of their encoded proteins. RNA molecules include, e.g.,small-interfering RNA (siRNA), short hairpin RNA (shRNA), anti-senseRNA, or micro RNA (miRNA). It is understood that item (ii) includes,e.g., replacement of an endogenous gene with a gene encoding a proteinhaving greater N-glycosylation activity relative to the endogenous geneso replaced. Genetic engineering also includes altering an endogenousgene encoding a protein having an N-glycosylation activity to produce aprotein having additions (e.g., a heterologous sequence), deletions, orsubstitutions (e.g., mutations such as point mutations; conservative ornon-conservative mutations). Mutations can be introduced specifically(e.g., site-directed mutagenesis or homologous recombination) or can beintroduced randomly (for example, cells can be chemically mutagenized asdescribed in, e.g., Newman and Ferro-Novick (1987) J. Cell Biol.105(4):1587.

The genetic modifications described herein can result in one or more of(i) an increase in one or more N-glycosylation activities in thegenetically modified cell, (ii) a decrease in one or moreN-glycosylation activities in the genetically modified cell, (iii) achange in the localization or intracellular distribution of one or moreN-glycosylation activities in the genetically modified cell, or (iv) achange in the ratio of one or more N-glycosylation activities in thegenetically modified cell. It is understood that an increase in theamount of an N-glycosylation activity can be due to overexpression ofone or more proteins having N-glycosylation activity, an increase incopy number of an endogenous gene (e.g., gene duplication), or analteration in the promoter or enhancer of an endogenous gene thatstimulates an increase in expression of the protein encoded by the gene.A decrease in one or more N-glycosylation activities can be due tooverexpression of a mutant form (e.g., a dominant negative form) of oneor more proteins having N-glysosylation altering activities,introduction or expression of one or more interfering RNA molecules thatreduce the expression of one or more proteins having an N-glycosylationactivity, or deletion of one or more endogenous genes that encode aprotein having N-glycosylation activity.

Methods of deleting or disrupting one or more endogenous genes aredescribed in the accompanying Examples. For example, to disrupt a geneby homologous recombination, a “gene replacement” vector can beconstructed in such a way to include a selectable marker gene. Theselectable marker gene can be operably linked, at both 5′ and 3′ end, toportions of the gene of sufficient length to mediate homologousrecombination. The selectable marker can be one of any number of geneswhich either complement host cell auxotrophy or provide antibioticresistance, including URA3, LEU2 and HIS3 genes. Other suitableselectable markers include the CAT gene, which confers chloramphenicolresistance to yeast cells, or the lacZ gene, which results in bluecolonies due to the expression of β-galactosidase. Linearized DNAfragments of the gene replacement vector are then introduced into thecells using methods well known in the art (see below). Integration ofthe linear fragments into the genome and the disruption of the gene canbe determined based on the selection marker and can be verified by, forexample, Southern blot analysis.

As detailed in the accompanying examples, subsequent to its use inselection, a selectable marker can be removed from the genome of thehost cell by, e.g., Cre-loxP systems (see below). This process of markerremoval is referred to as “curing” throughout the Examples.

Alternatively, a gene replacement vector can be constructed in such away as to include a portion of the gene to be disrupted, where theportion is devoid of any endogenous gene promoter sequence and encodesnone, or an inactive fragment of, the coding sequence of the gene. An“inactive fragment” is a fragment of the gene that encodes a proteinhaving, e.g., less than about 10% (e.g., less than about 9%, less thanabout 8%, less than about 7%, less than about 6%, less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, or 0%) of the activity of the protein produced from thefull-length coding sequence of the gene. Such a portion of the gene isinserted in a vector in such a way that no known promoter sequence isoperably linked to the gene sequence, but that a stop codon and atranscription termination sequence are operably linked to the portion ofthe gene sequence. This vector can be subsequently linearized in theportion of the gene sequence and transformed into a cell. By way ofsingle homologous recombination, this linearized vector is thenintegrated in the endogenous counterpart of the gene.

Expression vectors can be autonomous or integrative.

A recombinant nucleic acid can be in introduced into the cell in theform of an expression vector such as a plasmid, phage, transposon,cosmid or virus particle. The recombinant nucleic acid can be maintainedextrachromosomally or it can be integrated into the yeast cellchromosomal DNA. Expression vectors can contain selection marker genesencoding proteins required for cell viability under selected conditions(e.g., URA3, which encodes an enzyme necessary for uracil biosynthesisor TRP1, which encodes an enzyme required for tryptophan biosynthesis)to permit detection and/or selection of those cells transformed with thedesired nucleic acids (see, e.g., U.S. Pat. No. 4,704,362). Expressionvectors can also include an autonomous replication sequence (ARS). Forexample, U.S. Pat. No. 4,837,148 describes autonomous replicationsequences which provide a suitable means for maintaining plasmids inPichia pastoris.

Integrative vectors are disclosed, e.g., in U.S. Pat. No. 4,882,279.Integrative vectors generally include a serially arranged sequence of atleast a first insertable DNA fragment, a selectable marker gene, and asecond insertable DNA fragment. The first and second insertable DNAfragments are each about 200 (e.g., about 250, about 300, about 350,about 400, about 450, about 500, or about 1000 or more) nucleotides inlength and have nucleotide sequences which are homologous to portions ofthe genomic DNA of the species to be transformed. A nucleotide sequencecontaining a gene of interest (e.g., a gene encoding a protein havingN-glycosylation activity) for expression is inserted in this vectorbetween the first and second insertable DNA fragments whether before orafter the marker gene. Integrative vectors can be linearized prior toyeast transformation to facilitate the integration of the nucleotidesequence of interest into the host cell genome.

An expression vector can feature a recombinant nucleic acid under thecontrol of a yeast (e.g., Yarrowia lipolytica, Arxula adeninivorans, orother related dimorphic yeast species) promoter, which enables them tobe expressed in yeast. Suitable yeast promoters include the TEF1, HP4D,GAP, POX2, ADC1, TPI1, ADH2, POX, and Gal10 promter. See, e.g., Madzaket al., (2000) J. Mol. Microbiol. Biotechnol. 2:207-216; Guarente et al.(1982) Proc. Natl. Acad. Sci. USA 79(23):7410. Additional suitablepromoters are described in, e.g., Zhu and Zhang (1999) Bioinformatics15(7-8):608-611 and U.S. Pat. No. 6,265,185. Where the expression vectoris to be introduced into an animal cell, such as a mammalian cell, theexpression vector can feature a recombinant nucleic acid under thecontrol of an animal cell promoter suitable for expression in the hostcell of interest. Examples of mammalian promoters include the SV40 andcytomegalovirus (CMV) promoters.

A promoter can be constitutive or inducible (conditional). Aconstitutive promoter is understood to be a promoter whose expression isconstant under the standard culturing conditions. Inducible promotersare promoters that are responsive to one or more induction cues. Forexample, an inducible promoter can be chemically regulated (e.g., apromoter whose transcriptional activity is regulated by the presence orabsence of a chemical inducing agent such as an alcohol, tetracycline, asteroid, a metal, or other small molecule) or physically regulated(e.g., a promoter whose transcriptional activity is regulated by thepresence or absence of a physical inducer such as light or high or lowtemperatures). An inducible promoter can also be indirectly regulated byone or more transcription factors that are themselves directly regulatedby chemical or physical cues.

Genetic engineering of a cell also includes activating an endogenousgene (e.g., a gene encoding a protein having N-glycosylation activity)that is present in the host cell, but is normally not expressed in thecells or is not expressed at significant levels in the cells. Forexample, a regulatory sequence (e.g., a gene promoter or an enhancer) ofa endogenous gene can be modified such that the operably-linked codingsequence exhibits increased expression. Homologous recombination ortargeting can be used to replace or disable the regulatory regionnormally associated with the gene with a regulatory sequence whichcauses the gene to be expressed at levels higher than evident in thecorresponding non-genetically engineered cell, or causes the gene todisplay a pattern of regulation or induction that is different thanevident in the corresponding non-genetically engineered cell. Suitablemethods for introducing alterations of a regulatory sequence (e.g., apromoter or enhancer) of a gene are described in, e.g., U.S. ApplicationPublication No. 20030147868.

It is understood that other genetically engineered modifications alsocan be conditional. For example, a gene can be conditionally deletedusing, e.g., a site-specific DNA recombinase such as the Cre-loxP system(see, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173 and U.S.Application Publication No. 20060014264).

A recombinant nucleic acid can be introduced into a cell describedherein using a variety of methods such as the spheroplast technique orthe whole-cell lithium chloride yeast transformation method. Othermethods useful for transformation of plasmids or linear nucleic acidvectors into cells are described in, for example, U.S. Pat. No.4,929,555; Hinnen et al. (1978) Proc. Nat. Acad. Sci. USA 75:1929; Itoet al. (1983) J. Bacteriol. 153:163; U.S. Pat. No. 4,879,231; andSreekrishna et al. (1987) Gene 59:115. Electroporation and PEG1000 wholecell transformation procedures may also be used, as described by Creggand Russel, Methods in Molecular Biology: Pichia Protocols, Chapter 3,Humana Press, Totowa, N.J., pp. 27-39 (1998). Transfection of animalcells can feature, for example, the introduction of a vector to thecells using calcium phosphate, electroporation, heat shock, liposomes,or transfection reagents such as FUGENE® or LIPOFECTAMINE®, or bycontacting naked nucleic acid vectors with the cells in solution (see,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual SecondEdition vol. 1, 2 and 3. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y., USA, November 1989.

Transformed yeast cells can be selected for by using appropriatetechniques including, but not limited to, culturing auxotrophic cellsafter transformation in the absence of the biochemical product required(due to the cell's auxotrophy), selection for and detection of a newphenotype, or culturing in the presence of an antibiotic which is toxicto the yeast in the absence of a resistance gene contained in thetransformants. Transformants can also be selected and/or verified byintegration of the expression cassette into the genome, which can beassessed by, e.g., Southern blot or PCR analysis.

Prior to introducing the vectors into a target cell of interest, thevectors can be grown (e.g., amplified) in bacterial cells such asEscherichia coli (E. coli). The vector DNA can be isolated frombacterial cells by any of the methods known in the art which result inthe purification of vector DNA from the bacterial milieu. The purifiedvector DNA can be extracted extensively with phenol, chloroform, andether, to ensure that no E. coli proteins are present in the plasmid DNApreparation, since these proteins can be toxic to mammalian cells.

Genetic engineering, as described herein, can be used to express (e.g.,overexpress), introduce modifications into, or delete any number ofgenes encoding proteins having N-glycosylation activity. Such proteinsinclude, for example, OCH1, ALG3, α-1,3-glucosyltransferase, GnT I,mannosidase II, GnT II, glucosidase II, or Gal T. The genes encodingproteins having N-glycosylation activity can be from any speciescontaining such genes. Exemplary fungal species from which genesencoding proteins having N-glycosylation activity can be obtainedinclude, without limitation, Pichia anomala, Pichia bovis, Pichiacanadensis, Pichia carsonii, Pichia farinose, Pichia fermentans, Pichiafluxuum, Pichia membranaefaciens, Pichia membranaefaciens, Candidavalida, Candida albicans, Candida ascalaphidarum, Candida amphixiae,Candida Antarctica, Candida atlantica, Candida atmosphaerica, Candidablattae, Candida carpophila, Candida cerambycidarum, Candida chauliodes,Candida corydalis, Candida dosseyi, Candida dubliniensis, Candidaergatensis, Candida fructus, Candida glabrata, Candida fermentati,Candida guilliermondii, Candida haemulonii, Candida insectamens, Candidainsectorum, Candida intermedia, Candida jeffresii, Candida kefyr,Candida krusei, Candida lusitaniae, Candida lyxosophila, Candidamaltosa, Candida membranifaciens, Candida milleri, Candida oleophila,Candida oregonensis, Candida parapsilosis, Candida quercitrusa, Candidashehatea, Candida temnochilae, Candida tenuis, Candida tropicalis,Candida tsuchiyae, Candida sinolaborantium, Candida sojae, Candidaviswanathii, Candida utilis, Pichia membranaefaciens, Pichia silvestris,Pichia membranaefaciens, Pichia chodati, Pichia membranaefaciens, Pichiamenbranaefaciens, Pichia minuscule, Pichia pastoris, Pichiapseudopolymorpha, Pichia quercuum, Pichia robertsii, Pichia saitoi,Pichia silvestrisi, Pichia strasburgensis, Pichia terricola, Pichiavanriji, Pseudozyma Antarctica, Rhodosporidium toruloides, Rhodotorulaglutinis, Saccharomyces bayanus, Saccharomyces bayanus, Saccharomycesmomdshuricus, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomycescerevisiae, Saccharomyces bisporus, Saccharomyces chevalieri,Saccharomyces delbrueckii, Saccharomyces exiguous, Saccharomycesfermentati, Saccharomyces fragilis, Saccharomyces marxianus,Saccharomyces mellis, Saccharomyces rosei, Saccharomyces rouxii,Saccharomyces uvarum, Saccharomyces willianus, Saccharomycodes ludwigii,Saccharomycopsis capsularis, Saccharomycopsis fibuligera,Saccharomycopsis fibuligera, Endomyces hordei, Endomycopsis fobuligera.Saturnispora saitoi, Schizosaccharomyces octosporus, Schizosaccharomycespombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Torulasporadelbrueckii, Saccharomyces dairensis, Torulaspora delbrueckii,Torulaspora fermentati, Saccharomyces fermentati, Torulasporadelbrueckii, Torulaspora rosei, Saccharomyces rosei, Torulasporadelbrueckii, Saccharomyces rosei, Torulaspora delbrueckii, Saccharomycesdelbrueckii, Torulaspora delbrueckii, Saccharomyces delbrueckii,Zygosaccharomyces mongolicus, Dorulaspora globosa, Debaryomycesglobosus, Torulopsis globosa, Trichosporon cutaneum, Trigonopsisvariabilis, Williopsis californica, Williopsis saturnus,Zygosaccharomyces bisporus, Zygosaccharomyces bisporus, Debaryomycesdisporua. Saccharomyces bisporas, Zygosaccharomyces bisporus,Saccharomyces bisporus, Zygosaccharomyces mellis, Zygosaccharomycespriorianus, Zygosaccharomyces rouxiim, Zygosaccharomyces rouxii,Zygosaccharomyces barkeri, Saccharomyces rouxii, Zygosaccharomycesrouxii, Zygosaccharomyces major, Saccharomyces rousii, Pichia anomala,Pichia bovis, Pichia Canadensis, Pichia carsonii, Pichia farinose,Pichia fermentans, Pichia fluxuum, Pichia membranaefaciens, Pichiapseudopolymorpha, Pichia quercuum, Pichia robertsii, PseudozymaAntarctica, Rhodosporidium toruloides, Rhodosporidium toruloides,Rhodotorula glutinis, Saccharomyces bayanus, Saccharomyces bayanus,Saccharomyces bisporus, Saccharomyces cerevisiae, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces fermentati,Saccharomyces fagilis, Saccharomycodes ludwigii, Schizosaccharomycespombe, Schwanniomyces occidentalis, Torulaspora delbrueckii, Torulasporaglobosa, Trigonopsis variabilis, Williopsis californica, Williopsissaturnus, Zygosaccharomyces bisporus, Zygosaccharomyces mellis,Zygosaccharomyces rouxii, or any other fungi (e.g., yeast) known in theart or described herein. Exemplary lower eukaryotes also include variousspecies of Aspergillus including, but not limited to, Aspergilluscaesiellus, Aspergillus candidus, Aspergillus carneus, Aspergillusclavatus, Aspergillus deflectus, Aspergillus flavus, Aspergillusfumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger,Aspergillus ochraceus, Aspergillus oryzae, Aspergillus parasiticus,Aspergillus penicilloides, Aspergillus restrictus, Aspergillus sojae,Aspergillus sydowii, Aspergillus tamari, Aspergillus terreus,Aspergillus ustus, or Aspergillus versicolor. Exemplary protozoal generafrom which genes encoding proteins having N-glycosylation activity canbe obtained include, without limitation, Blastocrithidia, Crithidia,Endotrypanum, Herpetomonas, Leishmania, Leptomonas, Phytomonas,Trypanosoma (e.g., T. bruceii, T. gambiense, T. rhodesiense, and T.cruzi), and Wallaceina. For example, the gene encoding GnT I can beobtained from human (Swiss Protein Accession No. P26572), rat,Arabidopsis, mouse, or Drosophila; the gene encoding GntII can beobtained from human, rat (Swiss Protein Accession No. Q09326),Arabidopsis, or mouse; the gene encoding Man II can be obtained fromhuman, rat, Arabidopsis, mouse, Drosophila (Swiss Protein Accession No.Q24451); and the gene encoding GalT can be obtained from human (SwissProtein Accession No. P15291), rat, mouse, or bovine.

In some embodiments, a genetically engineered cell lacks the OCH1(GenBank Accession No: AJ563920) gene or gene product (mRNA or protein)thereof. In some embodiments, a genetically engineered cell lacks theALG3 (Genbank® Accession Nos: XM_503488, Genolevures Ref: YALIOE03190g)gene or gene product (mRNA or protein) thereof. In some embodiments, agenetically engineered cell expresses (e.g., overexpresses) anα-1,3-glucosyltransferase (e.g., ALG6, Genbank® Accession Nos:XM_502922, Genolevures Ref: YALIOD17028g) protein. In some embodiments,a genetically engineered cell expresses an α-1,2-mannosidase (e.g.,Genbank Accession No.: AF212153) protein. In some embodiments, agenetically engineered cell expresses a GlcNAc-transferase I (e.g.,Swiss Prot. Accession No. P26572) protein. In some embodiments, agenetically engineered cell expresses a mannosidase II protein orcatalytic domain thereof (e.g., Swiss Prot. Accession No. Q24451). Insome embodiments, a genetically engineered cell expresses agalactosyltransferase I protein or catalytic domain thereof (e.g., SwissProt. Accession No. P15291). In some embodiments, the geneticallyengineered cell expresses a GlcNAc-transferase II protein or catalyticdomain thereof (e.g., Swiss Prot. Accession No. Q09326). In someembodiments, the genetically engineered cell expresses an alpha or betasubunit (or both the alpha and the beta subunit) of a glucosidase IIsuch as the glucosidase II of Yarrowia lipolytica, Trypanosoma brucei orAspergillus niger. A genetically engineered cell can have anycombination of these modifications.

For example, in some embodiments, a genetically engineered cell can lackthe OCH1 gene and express an α-1,2-mannosidase, GlcNAc-transferase I,mannosidase II, and a galactosyltransferase I. In some embodiment, agenetically engineered cell can lack the ALG3 gene, and express anα-1,2-mannosidase, GlcNAc-transferase I, GlcNAc-transferase I, and agalactosyltransferase I. Such a genetically engineered cell further canexpress an α-1,3-glucosyltransferase and/or express alpha and betasubunits of a glucosidase II and/or lack the OCH1 gene.

One of more of such proteins can be fusion proteins that contain aheterologous targeting sequence. For example, the α-1,2-mannosidase canhave an HDEL endoplasmic reticulum (ER)-retention amino acid sequence(see Examples). It is understood that any protein having N-glycosylationactivity can be engineered into a fusion protein comprising an HDELsequence. Other proteins can have heterologous sequences that target theprotein to the Golgi apparatus. For example, the first 100 N-terminalamino acids encoded by the yeast Kre2p gene, the first 36 N-terminalamino acids (Swiss Prot. Accession No. P38069) encoded by the S.cerevisiae Mnn2 gene, or the first 46 N-terminal amino acids encoded bythe S. cerevisiae Mnn2p gene can be used to target proteins to theGolgi. As such, nucleic acids encoding a protein to be expressed in afungal cell can include a nucleotide sequence encoding a targetingsequence to target the encoded protein to an intracellular compartment.For example, the α-1,2-mannosidase can be targeted to the ER, while theGnT I, GnTII, mannosidase, and Gal T can be targeted to the Golgi.

In embodiments where a protein having N-glycosylation activity isderived from a cell that is of a different type (e.g., of a differentspecies) than the cell into which the protein is to be expressed, anucleic acid encoding the protein can be codon-optimized for expressionin the particular cell of interest. For example, a nucleic acid encodinga protein having N-glycosylation from Trypanosoma brucei can becodon-optimized for expression in a yeast cell such as Yarrowialipolytica. Such codon-optimization can be useful for increasingexpression of the protein in the cell of interest. Methods forcodon-optimizing a nucleic acid encoding a protein are known in the artand described in, e.g., Gao et al. (Biotechnol. Prog. (2004) 20(2):443-448), Kotula et al. (Nat. Biotechn. (1991) 9, 1386-1389), andBennetzen et al. (J. Biol. Chem. (1982) 257(6):2036-3031). Table 1 showsthe codon usage for Yarrowia lipolytica. Data was derived from 2,945,919codons present in 5,967 coding sequences. The contents of Table 1 wereobtained from a Codon Usage Database, which can be found at world wideweb at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=284591.

TABLE 1 Yarrowia lipolytica Codon Usage Table UUU 15.9(46804) CU21.8(64161) AU 6.8(20043) GU 6.1(17849) UUC 23.0(67672) CC 20.6(60695)AC 23.1(68146) GC 6.1(17903) UUA 1.8(5280) CA 7.8(22845) AA 0.8(2494) GA0.4(1148) UUG 10.4(30576) CG 15.4(45255) AG 0.8(2325) GG 12.1(35555) CUU13.2(38890) CU 17.4(51329) AU 9.6(28191) GU 6.0(17622) CUC 22.6(66461)CC 23.3(68633) AC 14.4(42490) GC 4.4(12915) CUA 5.3(15548) CA 6.9(20234)AA 9.8(28769) GA 21.7(63881) CUG 33.5(98823) CG 6.8(20042) AG32.1(94609) GG 7.7(22606) AUU 22.4(66134) CU 16.2(47842) AU 8.9(26184)GU 6.7(19861) AUC 24.4(71810) CC 25.6(75551) AC 31.3(92161) GC9.8(28855) AUA 2.2(6342) CA 10.5(30844) AA 12.4(36672) GA 8.4(24674) AUG22.6(66620) CG 8.5(25021) AG 46.5(136914) GG 2.4(7208) GUU 15.8(46530)CU 25.5(75193) AU 21.5(63259) GU 16.6(48902) GUC 21.5(63401) CC32.7(96219) AC 38.3(112759) GC 21.8(64272) GUA 4.0(11840) CA 11.2(32999)AA 18.8(55382) GA 20.9(61597) GUG 25.7(75765) CG 8.9(26190) AG46.2(136241) GG 4.4(12883) Tablefields are shown as [triplet][frequency: per thousand] ([number]).

In some embodiments, human proteins can be introduced into the cell andone or more endogenous yeast proteins having N-glycosylation activitycan be suppressed (e.g., deleted or mutated). Techniques for“humanizing” a fungal glycosylation pathway are described in, e.g., Choiet al. (2003) Proc. Natl. Acad. Sci. USA 100(9):5022-5027; Vervecken etal. (2004) Appl. Environ. Microb. 70(5):2639-2646; and Gerngross (2004)Nature Biotech. 22(11):1410-1414.

Where the genetic engineering involves, e.g., changes in the expressionof a protein or expression of an exogenous protein (including a mutantform of an endogenous protein), a variety of techniques can be used todetermine if the genetically engineered cells express the protein. Forexample, the presence of mRNA encoding the protein or the protein itselfcan be detected using, e.g., Northern Blot or RT-PCR analysis or WesternBlot analysis, respectively. The intracellular localization of a proteinhaving N-glycosylation activity can be analyzed by using a variety oftechniques, including subcellular fractionation and immunofluorescence.

Methods for detecting glycosylation of a target molecule include DNAsequencer-assisted (DSA), fluorophore-assisted carbohydrateelectrophoresis (FACE) or surface-enhanced laser desorption/ionizationtime-of-flight mass spectrometry (SELDI-TOF MS). For example, ananalysis can utilize DSA-FACE in which, for example, glycoproteins aredenatured followed by immobilization on, e.g., a membrane. Theglycoproteins can then be reduced with a suitable reducing agent such asdithiothreitol (DTT) or β-mercaptoethanol. The sulfhydryl groups of theproteins can be carboxylated using an acid such as iodoacetic acid.Next, the N-glycans can be released from the protein using an enzymesuch as N-glycosidase F. N-glycans, optionally, can be reconstituted andderivatized by reductive amination. The derivatized N-glycans can thenbe concentrated. Instrumentation suitable for N-glycan analysisincludes, e.g., the ABI PRISM® 377 DNA sequencer (Applied Biosystems).Data analysis can be performed using, e.g., GENESCAN® 3.1 software(Applied Biosystems). Optionally, isolated mannoproteins can be furthertreated with one or more enzymes to confirm their N-glycan status.Additional methods of N-glycan analysis include, e.g., mass spectrometry(e.g., MALDI-TOF-MS), high-pressure liquid chromatography (HPLC) onnormal phase, reversed phase and ion exchange chromatography (e.g., withpulsed amperometric detection when glycans are not labeled and with UVabsorbance or fluorescence if glycans are appropriately labeled). Seealso Callewaert et al. (2001) Glycobiology 11(4):275-281 and Freire etal. (2006) Bioconjug. Chem. 17(2):559-564.

Where any of the genetic modifications of the genetically engineeredcell are inducible or conditional on the presence of an inducing cue(e.g., a chemical or physical cue), the genetically engineered cell can,optionally, be cultured in the presence of an inducing agent before,during, or subsequent to the introduction of the nucleic acid. Forexample, following introduction of the nucleic acid encoding a targetprotein, the cell can be exposed to a chemical inducing agent that iscapable of promoting the expression of one or more proteins havingN-glycosylation activity. Where multiple inducing cues induceconditional expression of one or more proteins having N-glycosylationactivity, a cell can be contacted with multiple inducing agents.

Target molecules modified to include the desired N-glycan can beisolated from the genetically engineered cell. The modified targetmolecule can be maintained within the yeast cell and released upon celllysis or the modified target molecule can be secreted into the culturemedium via a mechanism provided by a coding sequence (either native tothe exogenous nucleic acid or engineered into the expression vector),which directs secretion of the molecule from the cell. The presence ofthe modified target molecule in the cell lysate or culture medium can beverified by a variety of standard protocols for detecting the presenceof the molecule. For example, where the altered target molecule is aprotein, such protocols can include, but are not limited to,immunoblotting or radioimmunoprecipitation with an antibody specific forthe altered target protein (or the target protein itself), binding of aligand specific for the altered target protein (or the target proteinitself), or testing for a specific enzyme activity of the modifiedtarget protein (or the target protein itself).

In some embodiments, at least about 25% of the target molecules isolatedfrom the genetically engineered cell contain the desired N-glycan. Forexample, at least about 27%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, or at least about 95%, or at least about 99% of the targetmolecules isolated from the genetically engineered cell can contain thedesired N-glycan.

In some embodiments, in the target molecules produced using the methodsdescribed herein, at least 50% (e.g., at least 55, 60, 65, 70, 75, 80,or 85%) of the N-glycans on the glycoprotein can be GlcNAc₂Man₃GlcNAc₂N-glycans. The percentage of GlcNAc₂Man₃GlcNAc₂ N-glycans can beestimated from the peak areas in the DSA-FACE electropherograms. SeeExample 13.

In some embodiments, the isolated modified target molecules can befrozen, lyophilized, or immobilized and stored under appropriateconditions, e.g., which allow the altered target molecules to retainbiological activity.

Cultures of Engineered Cells

This document also provides a substantially pure culture of any of thegenetically engineered cells described herein. As used herein, a“substantially pure culture” of a genetically engineered cell is aculture of that cell in which less than about 40% (i.e., less thanabout: 35%; 30%; 25%; 20%; 15%; 10%; 5%; 2%; 1%; 0.5%; 0.25%; 0.1%;0.01%; 0.001%; 0.0001%; or even less) of the total number of viablecells in the culture are viable cells other than the geneticallyengineered cell, e.g., bacterial, fungal (including yeast), mycoplasmal,or protozoan cells. The term “about” in this context means that therelevant percentage can be 15% percent of the specified percentage aboveor below the specified percentage. Thus, for example, about 20% can be17% to 23%. Such a culture of genetically engineered cells includes thecells and a growth, storage, or transport medium. Media can be liquid,semi-solid (e.g., gelatinous media), or frozen. The culture includes thecells growing in the liquid or in/on the semi-solid medium or beingstored or transported in a storage or transport medium, including afrozen storage or transport medium. The cultures are in a culture vesselor storage vessel or substrate (e.g., a culture dish, flask, or tube ora storage vial or tube).

The genetically engineered cells described herein can be stored, forexample, as frozen cell suspensions, e.g., in buffer containing acryoprotectant such as glycerol or sucrose, as lyophilized cells.Alternatively, they can be stored, for example, as dried cellpreparations obtained, e.g., by fluidized bed drying or spray drying, orany other suitable drying method.

Disorders Treatable by Altered N-Glycosylation Molecules

The isolated, target molecules modified to contain the desired N-glycancan be used to treat a variety of disorders, including metabolicdisorders, cancer, and inflammatory disorders.

(i) Metabolic Disorders

A metabolic disorder is one that affects the production of energy withinindividual human (or animal) cells. Most metabolic disorders aregenetic, though some can be “acquired” as a result of diet, toxins,infections, etc. Genetic metabolic disorders are also known as inbornerrors of metabolism. In general, the genetic metabolic disorders arecaused by genetic defects that result in missing or improperlyconstructed enzymes necessary for some step in the metabolic process ofthe cell. The largest classes of metabolic disorders are disorders ofcarbohydrate metabolism, disorders of amino acid metabolism, disordersof organic acid metabolism (organic acidurias), disorders of fatty acidoxidation and mitochondrial metabolism, disorders of porphyrinmetabolism, disorders of purine or pyrimidine metabolism, disorders ofsteroid metabolism disorders of mitochondrial function, disorders ofperoxisomal function, and lysosomal storage disorders (LSDs).

Examples of metabolic disorders that can be treated through theadministration of one or more glycosylated molecules (or pharmaceuticalcompositions of the same) described herein can include hereditaryhemochromatosis, oculocutaneous albinism, protein C deficiency, type Ihereditary angioedema, congenital sucrase-isomaltase deficiency,Crigler-Najjar type II, Laron syndrome, hereditary Myeloperoxidase,primary hypothyroidism, congenital long QT syndrome, tyroxine bindingglobulin deficiency, familial hypercholesterolemia, familialchylomicronemia, abeta-lipoproteinema, low plasma lipoprotein A levels,hereditary emphysema with liver injury, congenital hypothyroidism,osteogenesis imperfecta, hereditary hypofibrinogenemia,alpha-lantichymotrypsin deficiency, nephrogenic diabetes insipidus,neurohypophyseal diabetes insipidus, adenosine deaminase deficiency,Pelizaeus Merzbacher disease, von Willebrand disease type IIA, combinedfactors V and VIII deficiency, spondylo-epiphyseal dysplasia tarda,choroideremia, I cell disease, Batten disease, ataxia telangiectasias,ADPKD-autosomal dominant polycystic kidney disease, microvillusinclusion disease, tuberous sclerosis, oculocerebro-renal syndrome ofLowe, amyotrophic lateral sclerosis, myelodysplastic syndrome, Barelymphocyte syndrome, Tangier disease, familial intrahepatic cholestasis,X-linked adreno-leukodystrophy, Scott syndrome, Hermansky-Pudlaksyndrome types 1 and 2, Zellweger syndrome, rhizomelic chondrodysplasiapuncta, autosomal recessive primary hyperoxaluria, Mohr Tranebjaergsyndrome, spinal and bullar muscular atrophy, primary ciliary diskenesia(Kartagener's syndrome), giantism and acromegaly, galactorrhea,Addison's disease, adrenal virilism, Cushing's syndrome, ketoacidosis,primary or secondary aldosteronism, Miller Dieker syndrome,lissencephaly, motor neuron disease, Usher's syndrome, Wiskott-Aldrichsyndrome, Optiz syndrome, Huntington's disease, hereditary pancreatitis,anti-phospholipid syndrome, overlap connective tissue disease, Sjögren'ssyndrome, stiff-man syndrome, Brugada syndrome, congenital nephriticsyndrome of the Finnish type, Dubin-Johnson syndrome, X-linkedhypophosphosphatemia, Pendred syndrome, persistent hyperinsulinemichypoglycemia of infancy, hereditary spherocytosis, aceruloplasminemia,infantile neuronal ceroid lipofuscinosis, pseudoachondroplasia andmultiple epiphyseal, Stargardt-like macular dystrophy, X-linkedCharcot-Marie-Tooth disease, autosomal dominant retinitis pigmentosa,Wolcott-Rallison syndrome, Cushing's disease, limb-girdle musculardystrophy, mucoploy-saccharidosis type IV, hereditary familialamyloidosis of Finish, Anderson disease, sarcoma, chronic myelomonocyticleukemia, cardiomyopathy, faciogenital dysplasia, Torsion disease,Huntington and spinocerebellar ataxias, hereditary hyperhomosyteinemia,polyneuropathy, lower motor neuron disease, pigmented retinitis,seronegative polyarthritis, interstitial pulmonary fibrosis, Raynaud'sphenomenon, Wegner's granulomatosis, preoteinuria, CDG-Ia, CDG-Ib,CDG-Ic, CDG-Id, CDG-Ie, CDG-If, CDG-IIa, CDG-IIb, CDG-IIc, CDG-IId,Ehlers-Danlos syndrome, multiple exostoses, Griscelli syndrome (type 1or type 2), or X-linked non-specific mental retardation. In addition,metabolic disorders can also include lysosomal storage disorders suchas, but not limited to, Fabry disease, Farber disease, Gaucher disease,GM₁-gangliosidosis, Tay-Sachs disease, Sandhoff disease, GM₂ activatordisease, Krabbe disease, metachromatic leukodystrophy, Niemann-Pickdisease (types A, B, and C), Hurler disease, Scheie disease, Hunterdisease, Sanfilippo disease, Morquio disease, Maroteaux-Lamy disease,hyaluronidase deficiency, aspartylglucosaminuria, fucosidosis,mannosidosis, Schindler disease, sialidosis type 1, Pompe disease,Pycnodysostosis, ceroid lipofuscinosis, cholesterol ester storagedisease, Wolman disease, Multiple sulfatase deficiency,galactosialidosis, mucolipidosis (types II, III, and IV), cystinosis,sialic acid storage disorder, chylomicron retention disease withMarinesco-Sjögren syndrome, Hermansky-Pudlak syndrome, Chediak-Higashisyndrome, Danon disease, or Geleophysic dysplasia.

Symptoms of a metabolic disorder are numerous and diverse and caninclude one or more of, e.g., anemia, fatigue, bruising easily, lowblood platelets, liver enlargement, spleen enlargement, skeletalweakening, lung impairment, infections (e.g., chest infections orpneumonias), kidney impairment, progressive brain damage, seizures,extra thick meconium, coughing, wheezing, excess saliva or mucousproduction, shortness of breath, abdominal pain, occluded bowel or gut,fertility problems, polyps in the nose, clubbing of the finger/toe nailsand skin, pain in the hands or feet, angiokeratoma, decreasedperspiration, corneal and lenticular opacities, cataracts, mitral valveprolapse and/or regurgitation, cardiomegaly, temperature intolerance,difficulty walking, difficulty swallowing, progressive vision loss,progressive hearing loss, hypotonia, macroglossia, areflexia, lower backpain, sleep apnea, orthopnea, somnolence, lordosis, or scoliosis. It isunderstood that due to the diverse nature of the defective or absentproteins and the resulting disease phenotypes (e.g., symptomaticpresentation of a metabolic disorder), a given disorder will generallypresent only symptoms characteristic to that particular disorder. Forexample, a patient with Fabry disease can present a particular subset ofthe above-mentioned symptoms such as, but not limited to, temperatureintolerance, corneal whirling, pain, skin rashes, nausea, or dirarrhea.A patient with Gaucher syndrome can present with splenomegaly,cirrhosis, convulsions, hypertonia, apnea, osteoporosis, or skindiscoloration.

In addition to the administration of one or more molecules describedherein, a metabolic disorder can also be treated by proper nutrition andvitamins (e.g., cofactor therapy), physical therapy, and painmedications.

Depending on the specific nature of a given metabolic disorder, apatient can present these symptoms at any age. In many cases, symptomscan present in childhood or in early adulthood. For example, symptoms ofFabry disease can present at an early age, e.g., at 10 or 11 years ofage.

As used herein, a subject “at risk of developing a metabolic disorder”is a subject that has a predisposition to develop a disorder, i.e., agenetic predisposition to develop metabolic disorder as a result of amutation in a enzyme such as alpha-L-iduronidase, beta-D-galactosidase,beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase,alpha-L-fucosidase, arylsulfatase B, arylsulfatase A,alpha-N-acteylgalactosaminidase, aspartylglucosaminidase,iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase,beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase,alpha-neurominidase, phosphotransferase, acid lipase, acid ceramidase,sphinogmyelinase, thioesterase, cathepsin K, or lipoprotein lipase.Clearly, subjects “at risk of developing a metabolic disorder” are notall the subjects within a species of interest.

A subject “suspected of having a disorder” is one having one or moresymptoms of a disorder such as any of those described herein.

(ii) Cancer

Cancer is a class of diseases or disorders characterized by uncontrolleddivision of cells and the ability of these to spread, either by directgrowth into adjacent tissue through invasion, or by implantation intodistant sites by metastasis (where cancer cells are transported throughthe bloodstream or lymphatic system). Cancer can affect people at allages, but risk tends to increase with age. Types of cancers can include,e.g., lung cancer, breast cancer, colon cancer, pancreatic cancer, renalcancer, stomach cancer, liver cancer, bone cancer, hematological cancer,neural tissue cancer, melanoma, thyroid cancer, ovarian cancer,testicular cancer, prostate cancer, cervical cancer, vaginal cancer, orbladder cancer.

As used herein, a subject “at risk of developing a cancer” is a subjectthat has a predisposition to develop a cancer, i.e., a geneticpredisposition to develop cancer such as a mutation in a tumorsuppressor gene (e.g., mutation in BRCA1, p53, RB, or APC) or has beenexposed to conditions that can result in cancer. Thus, a subject canalso be one “at risk of developing a cancer” when the subject has beenexposed to mutagenic or carcinogenic levels of certain compounds (e.g.,carcinogenic compounds in cigarette smoke such as Acrolein, Arsenic,Benzene, Benz{a}anthracene, Benzo{a}pyrene, Polonium-210 (Radon),Urethane, or Vinyl Chloride). Moreover, the subject can be “at risk ofdeveloping a cancer” when the subject has been exposed to, e.g., largedoses of ultraviolet light or X-irradiation, or exposed (e.g., infected)to a tumor-causing/associated virus such as papillomavirus, Epstein-Barrvirus, hepatitis B virus, or human T-cell leukemia-lymphoma virus. Fromthe above it will be clear that subjects “at risk of developing acancer” are not all the subjects within a species of interest.

A subject “suspected of having a cancer” is one having one or moresymptoms of a cancer. Symptoms of cancer are well-known to those ofskill in the art and include, without limitation, breast lumps, nipplechanges, breast cysts, breast pain, weight loss, weakness, excessivefatigue, difficulty eating, loss of appetite, chronic cough, worseningbreathlessness, coughing up blood, blood in the urine, blood in stool,nausea, vomiting, liver metastases, lung metastases, bone metastases,abdominal fullness, bloating, fluid in peritoneal cavity, vaginalbleeding, constipation, abdominal distension, perforation of colon,acute peritonitis (infection, fever, pain), pain, vomiting blood, heavysweating, fever, high blood pressure, anemia, diarrhea, jaundice,dizziness, chills, muscle spasms, colon metastases, lung metastases,bladder metastases, liver metastases, bone metastases, kidneymetastases, and pancreas metastases, difficulty swallowing, and thelike. From the above it will be clear that subjects “suspected of havinga cancer” are not all the subjects within a species of interest.

In addition to the administration of one or more altered N-glycosylationmolecules described herein, a cancer can also be treated bychemotherapeutic agents, ionizing radiation, immunotherapy agents, orhyperthermotherapy agents. Chemotherapeutic agents include, e.g.,cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen,taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, andmethotrexate.

(iii) Inflammatory Disorders

An “inflammatory disorder,” as used herein, refers to a process in whichone or more substances (e.g., substances not naturally occurring in thesubject), via the action of white blood cells (e.g., B cells, T cells,macrophages, monocytes, or dendritic cells) inappropriately trigger apathological response, e.g., a pathological immune response.

Accordingly, such cells involved in the inflammatory response arereferred to as “inflammatory cells.” The inappropriately triggeredinflammatory response can be one where no foreign substance (e.g., anantigen, a virus, a bacterium, a fungus) is present in or on thesubject. The inappropriately triggered response can be one where aself-component (e.g., a self-antigen) is targeted (e.g., an autoimmunedisorder such as multiple sclerosis) by the inflammatory cells. Theinappropriately triggered response can also be a response that isinappropriate in magnitude or duration, e.g., anaphylaxis. Thus, theinappropriately targeted response can be due to the presence of amicrobial infection (e.g., viral, bacterial, or fungal). Types ofinflammatory disorders (e.g., autoimmune disease) can include, but arenot limited to, osteoarthritis, rheumatoid arthritis (RA),spondyloarthropathies, POEMS syndrome, Crohn's disease, multicentricCastleman's disease, systemic lupus erythematosus (SLE), multiplesclerosis (MS), muscular dystrophy (MD), insulin-dependent diabetesmellitus (IDDM), dermatomyositis, polymyositis, inflammatoryneuropathies such as Guillain Barre syndrome, vasculitis such asWegener's granulomatosus, polyarteritis nodosa, polymyalgia rheumatica,temporal arteritis, Sjogren's syndrome, Bechet's disease, Churg-Strausssyndrome, or Takayasu's arteritis. Also included in inflammatorydisorders are certain types of allergies such as rhinitis, sinusitis,urticaria, hives, angioedema, atopic dermatitis, food allergies (e.g., anut allergy), drug allergies (e.g., penicillin), insect allergies (e.g.,allergy to a bee sting), or mastocytosis. Inflammatory disorders canalso include ulcerative colitis and asthma.

A subject “at risk of developing an inflammatory disorder” refers to asubject with a family history of one or more inflammatory disorders(e.g., a genetic predisposition to one or more inflammatory disorders)or one exposed to one or more inflammation-inducing conditions. Forexample, a subject can have been exposed to a viral or bacterialsuperantigen such as, but not limited to, staphylococcal enterotoxins(SEs), a Streptococcus pyogenes exotoxin (SPE), a Staphylococcus aureustoxic shock-syndrome toxin (TSST-1), a streptococcal mitogenic exotoxin(SME) and a streptococcal superantigen (SSA). From the above it will beclear that subjects “at risk of developing an inflammatory disorder” arenot all the subjects within a species of interest.

A subject “suspected of having an inflammatory disorder” is one whopresents with one or more symptoms of an inflammatory disorder. Symptomsof inflammatory disorders are well known in the art and include, but arenot limited to, redness, swelling (e.g., swollen joints), joints thatare warm to the touch, joint pain, stiffness, loss of joint function,fever, chills, fatigue, loss of energy, headaches, loss of appetite,muscle stiffness, insomnia, itchiness, stuffy nose, sneezing, coughing,one or more neurologic symptoms such as dizziness, seizures, or pain.From the above it will be clear that subjects “suspected of having aninflammatory disorder” are not all the subjects within a species ofinterest.

In addition to the administration of one or more molecules describedherein, an inflammatory disorder can also be treated by non-steroidalanti-inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug(DMARD), a biological response modifier, or a corticosteroid. Biologicalresponse modifiers include, e.g., an anti-TNF agent. Non-limitingexamples of anti-TNF agents include a soluble TNF receptor or anantibody specific for TNF such as adulimumab, infliximab, or etanercept.

Methods suitable for treating (e.g., preventing or ameliorating one ormore symptoms of) any of the disorders described herein using any of thealtered N-glycosylation molecules (or pharmaceutical compositionsthereof) are set forth in the following section.

Pharmaceutical Compositions and Methods of Treatment

A target molecule modified to have the desired N-glycan can beincorporated into a pharmaceutical composition containing atherapeutically effective amount of the molecule and one or moreadjuvants, excipients, carriers, and/or diluents. Acceptable diluents,carriers and excipients typically do not adversely affect a recipient'shomeostasis (e.g., electrolyte balance). Acceptable carriers includebiocompatible, inert or bioabsorbable salts, buffering agents, oligo- orpolysaccharides, polymers, viscosity-improving agents, preservatives andthe like. One exemplary carrier is physiologic saline (0.15 M NaCl, pH7.0 to 7.4). Another exemplary carrier is 50 mM sodium phosphate, 100 mMsodium chloride. Further details on techniques for formulation andadministration of pharmaceutical compositions can be found in, e.g.,Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).Supplementary active compounds can also be incorporated into thecompositions.

Administration of a pharmaceutical composition containing molecules withN-glycans can be systemic or local. Pharmaceutical compositions can beformulated such that they are suitable for parenteral and/ornon-parenteral administration. Specific administration modalitiesinclude subcutaneous, intravenous, intramuscular, intraperitoneal,transdermal, intrathecal, oral, rectal, buccal, topical, nasal,ophthalmic, intra-articular, intra-arterial, sub-arachnoid, bronchial,lymphatic, vaginal, and intra-uterine administration.

Administration can be by periodic injections of a bolus of thepharmaceutical composition or can be uninterrupted or continuous byintravenous or intraperitoneal administration from a reservoir which isexternal (e.g., an IV bag) or internal (e.g., a bioerodable implant, abioartificial organ, or a colony of implanted altered N-glycosylationmolecule production cells). See, e.g., U.S. Pat. Nos. 4,407,957,5,798,113, and 5,800,828. Administration of a pharmaceutical compositioncan be achieved using suitable delivery means such as: a pump (see,e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer, 41:1270 (1993);Cancer Research, 44:1698 (1984); microencapsulation (see, e.g., U.S.Pat. Nos. 4,352,883; 4,353,888; and 5,084,350); continuous releasepolymer implants (see, e.g., Sabel, U.S. Pat. No. 4,883,666);macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881,4,976,859 and 4,968,733 and published PCT patent applicationsWO92/19195, WO 95/05452); injection, either subcutaneously,intravenously, intra-arterially, intramuscularly, or to other suitablesite; or oral administration, in capsule, liquid, tablet, pill, orprolonged release formulation.

Examples of parenteral delivery systems include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, pumpdelivery, encapsulated cell delivery, liposomal delivery,needle-delivered injection, needle-less injection, nebulizer,aerosolizer, electroporation, and transdermal patch.

Formulations suitable for parenteral administration conveniently containa sterile aqueous preparation of the altered N-glycosylation molecule,which preferably is isotonic with the blood of the recipient (e.g.,physiological saline solution). Formulations can be presented inunit-dose or multi-dose form.

Formulations suitable for oral administration can be presented asdiscrete units such as capsules, cachets, tablets, or lozenges, eachcontaining a predetermined amount of the altered N-glycosylationmolecule; or a suspension in an aqueous liquor or a non-aqueous liquid,such as a syrup, an elixir, an emulsion, or a draught.

A molecule having N-glycans suitable for topical administration can beadministered to a mammal (e.g., a human patient) as, e.g., a cream, aspray, a foam, a gel, an ointment, a salve, or a dry rub. A dry rub canbe rehydrated at the site of administration. Such molecules can also beinfused directly into (e.g., soaked into and dried) a bandage, gauze, orpatch, which can then be applied topically. Such molecules can also bemaintained in a semi-liquid, gelled, or fully-liquid state in a bandage,gauze, or patch for topical administration (see, e.g., U.S. Pat. No.4,307,717).

Therapeutically effective amounts of a pharmaceutical composition can beadministered to a subject in need thereof in a dosage regimenascertainable by one of skill in the art. For example, a composition canbe administered to the subject, e.g., systemically at a dosage from 0.01μg/kg to 10,000 μg/kg body weight of the subject, per dose. In anotherexample, the dosage is from 1 μg/kg to 100 μg/kg body weight of thesubject, per dose. In another example, the dosage is from 1 μg/kg to 30μg/kg body weight of the subject, per dose, e.g., from 3 μg/kg to 10μg/kg body weight of the subject, per dose.

In order to optimize therapeutic efficacy, a molecule containing anN-glycan can be first administered at different dosing regimens. Theunit dose and regimen depend on factors that include, e.g., the speciesof mammal, its immune status, the body weight of the mammal. Typically,levels of such a molecule in a tissue can be monitored using appropriatescreening assays as part of a clinical testing procedure, e.g., todetermine the efficacy of a given treatment regimen.

The frequency of dosing for a molecule is within the skills and clinicaljudgement of medical practitioners (e.g., doctors or nurses). Typically,the administration regime is established by clinical trials which mayestablish optimal administration parameters. However, the practitionermay vary such administration regimes according to the subject's age,health, weight, sex and medical status. The frequency of dosing can bevaried depending on whether the treatment is prophylactic ortherapeutic.

Toxicity and therapeutic efficacy of such molecules or pharmaceuticalcompositions thereof can be determined by known pharmaceuticalprocedures in, for example, cell cultures or experimental animals. Theseprocedures can be used, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Pharmaceutical compositions that exhibit hightherapeutic indices are preferred. While pharmaceutical compositionsthat exhibit toxic side effects can be used, care should be taken todesign a delivery system that targets such compounds to the site ofaffected tissue in order to minimize potential damage to normal cells(e.g., non-target cells) and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in appropriate subjects(e.g., human patients). The dosage of such pharmaceutical compositionslies generally within a range of circulating concentrations that includethe ED50 with little or no toxicity. The dosage may vary within thisrange depending upon the dosage form employed and the route ofadministration utilized. For a pharmaceutical composition used asdescribed herein (e.g., for treating a metabolic disorder in a subject),the therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the pharmaceutical composition which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

As defined herein, a “therapeutically effective amount” of a moleculecontaining an N-glycan is an amount of the molecule that is capable ofproducing a medically desirable result (e.g., amelioration of one ormore symptoms of a metabolic disorder) in a treated subject. Atherapeutically effective amount (i.e., an effective dosage) canincludes milligram or microgram amounts of the compound per kilogram ofsubject or sample weight (e.g., about 1 microgram per kilogram to about500 milligrams per kilogram, about 100 micrograms per kilogram to about5 milligrams per kilogram, or about 1 microgram per kilogram to about 50micrograms per kilogram).

The subject can be any mammal, e.g., a human (e.g., a human patient) ora non-human primate (e.g., chimpanzee, baboon, or monkey), a mouse, arat, a rabbit, a guinea pig, a gerbil, a hamster, a horse, a type oflivestock (e.g., cow, pig, sheep, or goat), a dog, a cat, or a whale.

A molecule or pharmaceutical composition thereof described herein can beadministered to a subject as a combination therapy with anothertreatment, e.g., a treatment for a metabolic disorder (e.g., a lysosomalstorage disorder). For example, the combination therapy can includeadministering to the subject (e.g., a human patient) one or moreadditional agents that provide a therapeutic benefit to the subject whohas, or is at risk of developing, (or suspected of having) a metabolicdisorder (e.g., a lysosomal storage disorder). Thus, the compound orpharmaceutical composition and the one or more additional agents can beadministered at the same time. Alternatively, the molecule can beadministered first and the one or more additional agents administeredsecond, or vice versa.

It will be appreciated that in instances where a previous therapy isparticularly toxic (e.g., a treatment for a metabolic disorder withsignificant side-effect profiles), administration of a moleculedescribed herein can be used to offset and/or lessen the amount of thepreviously therapy to a level sufficient to give the same or improvedtherapeutic benefit, but without the toxicity.

Any of the pharmaceutical compositions described herein can be includedin a container, pack, or dispenser together with instructions foradministration.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES

Table 2 contains a list of all of the strains used in the experimentsdescribed below. In Table 2, MH=HDEL-tagged α-1,2-mannosidase; ζ=randomintegration via zeta sequences; docking Δ=integration into a specificlocus; and (H)=hygromycin resistant.

TABLE 2 Listing of Strains Used in Examples Number Short nameDescription Markers Expected N-Glycans G013 Po1d lnuga Δoch1(URA3) Po1dlnuga transformed with URA3⁺ leu2⁻ Mainly Man₈GlcNAc₂ cl 26.1SpeI/Bst1107I- digested ade2⁻ gut2⁻ pYLOCH1PUT-TOPO G014 Po1d lnugaΔoch1 (cured) Po1d lnuga Δoch1 (G013) ura3⁻ leu2⁻ Mainly Man₈GlcNAc₂ cl7 cured from the URA3 marker ade2⁻ gut2⁻ using pUB4-Cre G016 Po1d lnugaΔoch1 TefMH Po1d lnuga Δoch1 (cured) URA3⁺ leu2⁻ Man₅GlcNAc₂ (ζ-Not) cl1.4 (G014) transformed with NotI- ade2⁻ gut2⁻ digestedpYLTUXL2preManHDEL(Yl) G018 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1(cured) URA3⁺ leu2⁻ Man₅GlcNAc₂ (ζ-Not) cl 11.2 (G014) transformed withNotI- ade2⁻ gut2⁻ digested pYLHUXL2preManHDEL(Yl) G036 Po1d lnuga Δoch1Hp4dMH Po1d lnuga Δoch1 Hp4dMH ura3⁻ leu2⁻ Man₅GlcNAc₂ (ζ-Not)(cured) cl2.2 (Not) cl 11.2 (G018) cured ade2⁻ gut2⁻ (1 copy ManHDEL) from theURA3 marker using pRRQ2 G039 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1Hp4dMH URA3⁺ leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6(ζ-Not-cured) cl 2.2 (G036) ade2⁻ gut2⁻ and Man₃GlcNAc₂ cl 24.1transformed with NotI/PacI- digested pYlALG3PUT-ALG6 G040 Po1d lnugaΔoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ leu2⁻ GlcNAcMan₅GlcNAc₂(ζ-cured) TefhGnTI cl 5.4 (ζ-Not-cured) cl 2.2 (G036) ade2⁻ gut2⁻ (1copy GnT I) transformed with NotI- digested pYLTmAx hGnTI G043 Po1dlnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ LEU2⁺ GlcNAcMan₃GlcNAc₂(ζ-cured) TefhGnTI (ζ-cured) TefhGnTI cl 5.4 ade2⁻ gut2⁻ TefManII cl 15(G040) transformed with NotI- digested pYLTmAXDmManII (LEU2 ex) G044Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ leu2⁻GalGlcNAcMan₅ (ζ-cured) TefhGnTI (ζ-cured) TefhGnTI cl 5.4 ADE2⁺ gut2⁻GlcNAc₂ TefGalTI cl 12 (G040) transformed with NotI- digestedpYLTmAXSpGal10hGalTI (ADE2 ex) G045 Po1d lnuga Δoch1 Hp4dMH Po1d lnugaΔoch1 Hp4dMH ura3⁻ leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6(ζ-cured) Δalg3ALG6 cl 24.1 ade2⁻ gut2⁻ and Man₃GlcNAc₂ cl 2.16 (cured)(G039) cured from the URA3 marker using pRRQ2 G046 Po1d lnuga Δoch1Hp4dMH Po1d lnuga Δoch1 (cured) ura3⁻ leu2⁻ Man₅GlcNAc₂ (docking Δleu2)(G014) transformed with NotI- ADE2⁺ gut2⁻ digested JME926 pPTleu2-ADE2Ex- Hp4dManHDEL(Yl) G047 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1Hp4dMH URA3⁺ leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured)Δalg3ALG6 cl 24.1 ade2⁻ gut2⁻ and TefhGnTI(H) cl2 (G039) transformedwith Hyg^(R) GlcNAcMan₃GlcNAc₂ (1 copy GnT I) NotI-digested pYLTmAXhGnTI(Hyg^(R) ex) G048 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured) Δalg3ALG6 cl2.16 ade2⁻ gut2⁻ and (cured) TefhGnTI clone 7.3 (cured) (G045)transformed GlcNAcMan₃GlcNAc₂ with NotI-digested pYLTmAXhGnTI G050 Po1dlnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ leu2⁻Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured) Δalg3ALG6 cl 2.16ADE2⁺ gut2⁻ and (cured) TefhGnTI TefrGnTII (cured) (G045) transformedGlcNAc₂Man₃GlcNAc₂ cl. 42.3 with NotI-digested pYLTmAXhGnTI andpYLTmAXrGnTII (ADE2 Ex) G051 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1Hp4dMH URA3⁺ leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured)Δalg3ALG6 ADE2⁺ gut2⁻ and TefhGnTI(H) TefrGnTII TefhGnTI cl2 (G047)Hyg^(R) GlcNAc₂Man₃GlcNAc₂ clone 4.5 transformed with NotI- digestedpYLTmAXrGnTII (ADE2 Ex) G052 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1Hp4dMH URA3⁺ leu2⁻ GlcNAcMan₅GlcNAc₂ (ζ-cured) Hp4dhGnTI (ζ)(ζ-Not-cured) cl 2.2 (G036) ade2⁻ gut2⁻ cl one 16 transformed with NotI-digested pYLHp4mAxhGnTI G053 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1(cured) ura3⁻ LEU2⁺ Man₅GlcNAc₂ (docking Δaxp1) (G014) transformed withNotI- ade2⁻ gut2⁻ digested OXYP289- pPTAxp1-Leu2Ex- Hp4dManHDEL(Yl) G054Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH ura3⁻ leu2⁻ Man₅GlcNAc₂(docking Δaxp1) (cured) (docking Δaxp1) (G053) cured ade2⁻ gut2⁻ fromthe LEU2 marker using pUB4-Cre G055 Po1d lnuga Δoch1 Hp4dMH Po1d lnugaΔoch1 Hp4dMH ura3⁻ leu2⁻ Man₅GlcNAc₂ (docking Δleu2-cured) (dockingΔleu2) (G046) cured ade2⁻ gut2⁻ from the ADE2 marker using pRRQ2 G056Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ leu2⁻Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured) Δalg3ALG6 cl 2.16ade2⁻ gut2⁻ and Hp4dGnTI (ζ) clone E (cured) (G045) transformedGlcNAcMan₃GlcNAc₂ with NotI-digested pYLHp4mAxhGnTI G057 Po1d lnugaΔoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂(ζ-cured) Δalg3ALG6 (ζ-cured) Δalg3ALG6 cl 2.16 ade2⁻ gut2⁻ and Hp4dGnTI(docking Δade2) (cured) (G045) transformed GlcNAcMan₃GlcNAc₂ clone Gwith NotI-digested JME925 pPTade2-URA3ex- Hp4dhGnTI G058 Po1d lnugaΔoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH ura3⁻ leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂(ζ-cured) Δalg3ALG6 (ζ-cured) Δalg3ALG6 ade2⁻ gut2⁻ and Hp4dGnTI(ζ-cured) Hp4dGnTI (ζ) (G056) cured GlcNAcMan₃GlcNAc₂ from the URA3marker using pRRQ2 G059 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMHura3⁻ leu2⁻ Glc₁₋₂Man_(5′)GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured)Δalg3ALG6 ade2⁻ gut2⁻ and Hp4dGnTI (docking Δade2- Hp4dGnTI (dockingΔade2) GlcNAcMan₃GlcNAc₂ cured) (G057) cured from the URA3 marker usingpRRQ2 G060 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ LEU2⁺GlcNAcMan₃GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured) Δalg3ALG6 ade2− gut2⁻Hp4dGnTI (docking Δade2) Hp4dGnTI (docking Δade2) Hp4dGls2α/β (ζ) (G057)transformed with Not- clone 6 digested Zeta-LEU2Ex- Hp4dL2preAnGlcII a +b(alt) G061 Po1d lnuga Δoch1 Hp4dMH Po1d lnuga Δoch1 Hp4dMH URA3⁺ LEU2⁺GlcNAcMan₃GlcNAc₂ (ζ-cured) Δalg3ALG6 (ζ-cured) Δalg3ALG6 ade2− gut2−Hp4dGnTI (docking Δade2) Hp4dGnTI (docking Δade2) Hp4d Gls2α/β (docking(G057) transformed with Not- Δura3) clone 18 digested JME923 pPTUra3-Leu2Ex-Hp4d L2preAnGlcIIa + b (alt) G070 Po1d lnuga Δoch1 Hp4dMH Po1dlnuga Δoch1 Hp4dMH (1 URA3⁺ LEU2⁺ GlcNAc₂Man₃GlcNAc₂ (1 copy-ζ-cured)Δalg3ALG6 copy-ζ-cured) Δalg3ALG6 ADE2⁺ gut2⁻ (cured) Hp4dGnTI (docking(cured) Hp4dGnTI (docking Δade2) Hp4dGls2α/β Δade2) Hp4d Gls2α/β(docking Δura3) Hp4dGnTII (docking Δura3) (G061) (ζ) cl 6 transformedwith Not-digested pYLHp4mAXrGnTII (ADE2ex) G071 Po1d lnuga Δoch1 Hp4dMHPo1d lnuga Δoch1 Hp4dMH (1 URA3⁺ LEU2⁺ GlcNAc₂Man₃GlcNAc₂ (1copy-ζ-cured) Δalg3ALG6 copy-ζ-cured) Δalg3ALG6 ADE2⁺ gut2⁻ (cured)Hp4dGnTI (docking (cured) Hp4dGnTI (docking Δade2) Hp4d Gls2α/β Δade2)Hp4d Gls2α/β (docking Δura3) Hp4dGnTII (docking Δura3) (G061)(integration in Axp1 locus was transformed with Not-digested aimed at)cl 8 OXYP289 pPTAxp1-ADE2ex- Hp4dGnTII G096 Po1d lnuga Δoch1 Hp4dMH Po1dlnuga Δoch1 Hp4dMH (1 URA3⁺ LEU2⁺ GlcNAc₂Man₃GlcNAc₂ (1 copy-ζ-cured)Δalg3ALG6 copy-ζ-cured) Δalg3ALG6 ADE2⁺ GUT2⁺ (cured) Hp4dGnTI (docking(cured) Hp4dGnTI (docking Δade2) Hp4d Gls2α/β Δade2) Hp4d Gls2α/β(docking Δura3) Hp4dGnTII (docking Δura3) Hp4dGnTII (integration in Axp1locus was (docking Δaxp1) (G071) aimed at) Hp4dPP-HC/LC transformed withNotI digested clone 13 pYLHp4L2preproHerHC&LC (Gut2ex)-ori2

Example 1: Yarrowia lipolytica OCH1 Disruption

The generation of a glyco-engineered protein expression strain was donein Yarrowia lipolytica strain po1d lnuga (a strain having theauxotrophies leu2-, ura3-, gut2- and ade2-). A strategy to knock out theOCH1 (GenBank Accession No: AJ563920) gene in Yarrowia lipolytica wasset up as described for the LIP2 gene (Fickers et al., 2003 J MicrobiolMethods. 55(3):727-37). The gene construction strategy followed for theOCH1 gene is described in U.S. Patent Publication No. 20090069232-A1.The resulting vector was called pYlOCH1 PUT TOPO (FIG. 1B).

The OCH1 KO fragment was isolated from the plasmid by a SpeI/Bst1107Irestriction digest and transformed to Yarrowia lipolytica strain po1dlnuga. Several uracil prototrophic strains were obtained and screened byPCR on genomic DNA (gDNA) using primers Yloch1 prom fw(5′-TCGCTATCACGTCTCTAGC-3′, SEQ ID NO:1) and Yloch1 term rev(5′-ACTCTGTATACTTGTATGTACTGTGAGAC-3′, SEQ ID NO:2) to analyze thegenomic integration of the plasmid. A fragment of the correct size(i.e., 2328 bp vs. 1894 bp in the wild type) was amplified for severalclones tested. The knock-out of the OCH1 gene also was confirmed byN-glycan analysis of the total glycoprotein pool secreted into thegrowth medium (=secretome): the Man₅GlcNAc₂ structure has become thepredominant N-glycan within the sugar profile (FIG. 2). This profilediffers from that of the wild-type strain, which contains a higheramount of Man₉GlcNAc₂—the latter most probably containing an additionalmannose as a result of Och1p activity—as well as some structures with aneven higher number of mannose residues.

To remove the URA3 gene, a positive Δoch1 clone (called G013, see Table2) was transformed with the episomal plasmid pUB4-Cre (Fickers et al.,2003, supra) that contains an expression cassette for the Crerecombinase. Removal of the URA3 gene was screened for by PCR on gDNAusing primers Yloch1 prom fw and Yloch1 term rev (see above). Clones inwhich the URA3 marker was excised no longer resulted in theamplification of a 2328 bp band; instead a PCR-fragment of 1075 bp(excl. URA3) was obtained. Positive clones were checked at the N-glycanlevel of the secretome and show a profile very similar to that of thenon-cured strain (FIG. 2). One of the cured strains (called G014, seeTable 2) was selected for further N-glycan engineering.

Example 2: Overexpression of an ER-Retained α-1,2-Mannosidase by EitherRandom Integration or Targeted/Docked Integration

To enable the generation of Man₅GlcNAc₂ attached to glycoproteinsexpressed by a Δoch1 strain, an α-1,2-mannosidase was expressed tocleave Man₅GlcNAc₂ to Man₅GlcNAc₂ (i.e., a Golgi type α-1,2-mannosidaseactivity). Such a mannosidase should be targeted to the secretionsystem. Trichoderma reesei α-1,2-mannosidase (Genbank accession no.AF212153), fused to the S. cerevisiae prepro mating factor and taggedwith a HDEL sequence (SEQ ID NO:21) to localize it into the ER, is ableto trim Man₅GlcNAc₂ to Man₅GlcNAc₂ in vivo in Pichia pastoris as well asin Trichoderma reesei and Aspergillus niger. Expression constructs weremade where a codon-optimized version of the HDEL-tagged T. reeseiα-1,2-mannosidase was fused to the Y. lipolytica LIP2 pre signalsequence and placed under the transcriptional control of either theTEF1, Hp4d (Madzak et al., 2000, J. Mol. Microbiol. Biotechnol.2:207-216), GAP or POX2 promotor. The construction strategy of theseplasmids is described in U.S. Patent Publication No. 20090069232-A1.

Two of these vectors, pYLHUXdL2preManHDEL and pYLTUXdL2preManHDEL (FIG.3)—with the mannosidase under the transcriptional control of the Hp4dresp. TEF1 promotor, were used to transform strain G0014 (derived fromExample 1). The vectors were digested with NotI to allow randomintegration into the genome via the zeta sequences. URA3 prototrophictransformants were selected for N-glycan analysis. Several transformantsshow a clear conversion of Man₅GlcNAc₂ towards Man₅GlcNAc₂ (FIG. 4).Since clones expressing the mannosidase under TEF1 promotor controlshowed a slow and clumpy growth phenotype (one of these clones wascalled G016), further steps in glyco-engineering were done in a strainbackground where the gene is under Hp4d transcriptional control.

One positive clone expressing the ManHDEL under control of the hp4dpromoter (G018) was chosen, from which the URA3 marker was cured viatransient transformation of plasmid pRRQ2 (Richard et al., 2001 J.Bacteriol. 183:3098-3107), expressing the Cre-recombinase. Severalura3-clones were selected after the procedure and one clone (G036),showing a clear Man₅GlcNAc₂ profile on the secretome, was used forfurther engineering work (FIG. 4). Southern analysis of this clonerevealed the presence of one randomly integrated mannosidase expressioncassette. This Southern analysis was performed on Hind III digestedgenomic DNA using a DIG-labeled mannosidase-specific PCR fragment thatwas generated using primers Man for(5′-GCCTTCCAGACCTCTTGGAACGCCTACCACC-3′, SEQ ID NO:22) and Man rev(5′-GCCAGGTGGCCGCCTCGTCGAGAAGAAGATCG-3′, SEQ ID NO:23).

In an alternative strategy, two constructs were generated that allowtargeted integration of the Hp4d-driven mannosidase expression cassetteinto either the LEU2 or AXP1 locus of the Yarrowia genome. Constructionof these plasmids, JME926_pPTLeu2-ADE2ex-Hp4dManHDEL(Yl) and OXYP289pPTAxp1-LEU2ex-Hp4dManHDEL(Yl), is described in FIG. 5. Prior totransformation to strain G014, both constructs were digested with NotIand the respective expression cassettes were isolated. Selected ADE2prototrophic clones had potentially integrated the mannosidaseexpression cassette into the LEU2 locus, whereas LEU2 prototrophspotentially had integrated the cassette into the AXP1 locus. Thetransformants were checked by Southern analysis to assess propertargeting into the genome. This was performed on BamHI digested(integration in LEU2 locus) or HindIII digested (integration in AXP1locus) genomic DNA using a DIG-labeled mannosidase-specific PCR fragmentthat was generated using primers Man for(5′-GCCTTCCAGACCTCTTGGAACGCCTACCACC-3′, SEQ ID NO:22) and Man rev(5′-GCCAGGTGGCCGCCTCGTCGAGAAGAAGATCG-3′, SEQ ID NO:23). The selectedclones also were checked for the nature of the N-glycans synthesizedonto the secreted glycoproteins. In most cases, correctly targetedHp4d-driven α-1,2-mannosidase expression resulted into the synthesis ofpredominantly Man₅GlcNAc₂ oligosaccharides (FIG. 6). For each targetinglocus, one mannosidase expressing clone (G046 in case of LEU2 docking;G053 in case of AXP1 docking) was selected for curing via transientexpression of the Cre recombinase using plasmid pRRQ2 for strain G046and pUB4-Cre for strain G053. The resulting cured strains (G055 abdG054, respectively) were re-checked via Southern blotting and theirMan₅GlcNAc₂ profile confirmed via N-glycan analysis using DSA-FACE.

Example 3: Expression of GlcNAc-Transferase I

A Yarrowia codon-optimized sequence was generated for the expression ofa fusion protein consisting of the first 100 N-terminal amino acids ofthe S. cerevisiae Kre2 protein (SwissProt AccNo P27809) followed by thecatalytic domain of human GlcNAc-transferase I (SwissProt AccNoP26572)(FIG. 7, SEQ ID NO:3 and SEQ ID NO:4). The yeast Kre2p 100N-terminal amino acids serve as a Golgi localization signal for thecatalytic GnT I domain. In this way, it is ensured that the GnT I fusionprotein is localized later in the secretion pathway than the ER-retainedHDEL-tagged α-1,2-mannosidase in order to enable the enzyme convertingthe protein-linked N-glycans from Man₅GlcNAc₂ to GlcNAcMan₅GlcNAc₂. Thecodon optimized synthetic gene for the expression of the fusion proteinwas placed under the transcriptional control of either the TEF1 or theHp4d promoter, resulting into the plasmids pYLTmAXhGnTI andpYLHp4mAXhGnTI. The construction strategy is shown in FIG. 8. Functionalexpression of the Kre2-GnT I fusion protein should result in theaddition of a β-1,2-linked GlcNAc residue onto the available Man₅GlcNAc₂glycans resulting in the synthesis of GlcNAcMan₅GlcNAc₂.

The plasmids pYLTmAXhGnTI and pYLHp4mAXGnTI were NotI digested beforetransformation to strain G036 (cf. Example 2), known to produceMan₅GlcNAc₂ N-glycans on its secreted proteins. Transformants wereselected for uracil prototrophy. Analysis of the N-glycosylation profileon the secretome of several of these clones showed a clear change in theN-glycan pattern: the Man₅GlcNAc₂ was significantly reduced and a newpeak, representing an N-glycan with higher molecular weight (about oneglucose unit extra), appeared. Treatment of the isolated N-glycans withJack Bean (3-N-acetylhexosaminidase, an enzyme capable of removingterminal β-linked GlcNAc residues, indicated that the new N-glycan isGlcNAcMan₅GlcNAc₂: the new peak disappeared and was completely convertedinto Man₅GlcNAc₂ (FIG. 9). Depending on the cultivation method used,about 70% of the total N-glycan pool proved to be GlcNAcMan₅GlcNAc₂(with approximately 77% of the available Man₅GlcNAc₂ being converted).

One transformant expressing the Kre2-GnT I fusion protein under controlof the TEF1 promotor was named strain G040 and selected for further use.Genomic analysis of this strain via Southern blot indicated the presenceof one expression cassette. Southern analysis was done on BamHI digestedgenomic DNA using a DIG-labeled GnT I-specific PCR fragment that wasgenerated using primers 5′-GGATGATCACACAATGGCCCTGTTTCTG-3′ (SEQ ID NO:5)and 5′-TGCTCTAGACTAGTTCCAAGAGGGGTC-3′ (SEQ ID NO:6). Analysis of theglycosylation profile on the secretome of strain G040 versus strainscarrying 1 to 3 copies (confirmed by the same southern blot) of theHp4d-driven Kre2-GnT I expression cassette, did not show significantdifference in GlcNAc-transfer capacity.

Example 4: Expression of Mannosidase II

A Yarrowia codon-optimized sequence was generated for the expression ofa fusion protein consisting of the first 36 N-terminal amino acids ofthe S. cerevisiae Mnn2 protein (SwissProt AccNo P38069) followed by thecatalytic domain of Drosophila melanogaster mannosidase II (SwissProtAccNo Q24451)(FIG. 10, SEQ ID NO:7 and SEQ ID NO:8). The yeast Mnn2 36N-terminal amino acids serve as a Golgi localization signal for thecatalytic Man II domain. In this way, it is ensured that the Mnn2-Man IIfusion protein is localized at the same or even a later position in thesecretion pathway than the Kre2-GnT I fusion protein and is thereforeable to convert GlcNAcMan₅GlcNAc₂ into GlcNAcMan₃GlcNAc₂. The Yarrowiacodon optimized synthetic gene for the expression of the fusion proteinwas placed under the transcriptional control of the TEF1 promoter,resulting into the plasmids pYLTmAXDmManII and pYLTmAXDmManII (LEU2ex).The construction strategy is shown in FIG. 11.

Plasmid pYLTmAXDmManII (LEU2ex) was NotI digested before transformationto strain G040 (see Example 3), which was known to produceGlcNAcMan₅GlcNAc₂ N-glycans on its secreted proteins. Transformants wereselected for leucine prototrophy. Analysis of the N-glycosylationprofile on the secretome of several of these clones showed a change inthe N-glycan pattern: a new peak representing an N-glycan with a lowermolecular weight of about two glucose units appeared, which couldindicate the formation of GlcNAcMan₃GlcNAc₂ and thus partial mannosidaseII activity. Also another peak appears, running at almost the sameposition as Man₅GlcNAc₂ (i.e. a shoulder to the peak), potentiallyrepresenting GlcNAcMan₄GlcNAc₂. The latter structure could be the resultof a partial trimming event, where the mannosidase II activity has onlyremoved one mannose residue instead of two. Treatment of the isolatedN-glycans with Jack Bean β-N-acetylhexosaminidase resulted in a leftwardshift of the glycan pattern with about one glucose unit and thus ahigher electrophoretic mobility due to the loss of a terminal GlcNAcresidue (FIG. 12). This further confirms the generation ofGlcNAcMan₄GlcNAc₂ and GlcNAcMan₃GlcNAc₂ from GlcNAcMan₅GlcNAc₂ due tothe expression of a functional mannosidase II activity. Depending on thecultivation method used, about 15% of the total N-glycan pool proved tobe GlcNAcMan₃GlcNAc₂: approximately 35% of the availableGlcNAcMan₅GlcNAc₂ lost 1 or 2 mannose residues, with 20% beingcompletely trimmed towards GlcNAcMan₃GlcNAc₂.

Example 5: Expression of Galactosvltransferase I

Synthesis of N-glycans with terminal galactose residues not only dependson the presence of a functional and well-localized galactosyltransferasewithin the secretion pathway, but also on the availability of UDP-Gal,the donor substrate that is used by the enzyme. Although UDP-Glc andUDP-GlcNAc are generally thought to be sufficiently available in theGolgi apparatus of yeast organisms, this is less known for UDP-Gal. Toovercome potential UDP-Gal deficiency during glyco-engineering, attemptshave been made previously in Pichia pastoris to target a fusion proteinof the Schizosaccharomyces pombe UDP-Glc-4-epimerase (encoded by theGAL10 like gene SPBC365.14c—SwissProt AccNo Q9Y7X5) and the catalyticdomain of the human β-1,4-galactosyltransferase I (GalT I) (SwissProtAccNo P15291) into the yeast Golgi apparatus (Bobrowicz et al.,Glycobiology 14(9):757-766, 2004). Localization of the Gal10p-GalT Ifusion protein within the secretion pathway, preferably at a positionwhere GlcNAc-transfer and mannosidase II activity has already acted onthe N-glycans of proteins destined for secretion, was accomplished byusing the first 46 N-terminal amino acids of S. cerevisiae Mnn2p asN-terminal targeting signal.

Hence, a Yarrowia codon-optimized sequence was generated for theexpression of a fusion protein consisting of the first 46 N-terminalamino acids of the S. cerevisiae Mnn2 protein, followed by the S. pombeGal10-like protein and the catalytic domain of human GalT I (FIG. 13).The resulting synthetic gene was placed under the transcriptionalcontrol of the TEF1 promoter, resulting into the plasmidspYLTmAXSpGal10hGalTI and pYLTmAXSpGal10hGalTI (ADE2ex). The constructionstrategy is shown in FIG. 14.

Plasmid pYLTmAXSpGal10hGalTI (ADE2ex) was NotI digested beforetransformation to strain G040 (see Example 3), known to produceGlcNAcMan₅GlcNAc₂ N-glycans on its secreted proteins. Transformants wereselected for their adenine prototrophy. Analysis of the N-glycosylationprofile on the secretome of several of these clones showed a change inthe N-glycan pattern: a new peak appears, running at a position betweenMan₇GlcNAc₂ and Man₅GlcNAc₂ (FIG. 15). Treatment of the N-glycans withStreptococcus pneumonia β-1,4-galactosidase indicates that the peakrepresents GalGlcNAcMan₅GlcNAc₂ since this in vitro digest results inthe disappearance of this new peak and an equally high increase inGlcNAcMan₅GlcNAc₂.

Using this set-up and depending on the growth conditions, about 75% ofGlcNAcMan₅GlcNAc₂ was converted into GalGlcNAcMan₅GlcNAc₂. The totalamount of the galactosylated structure accounted for about 25% of thetotal N-glycan pool. From an in vitro α-1,2-mannosidase digest it isclear, however, that a significant amount of high-mannose N-glycans wasnot converted to Man₅GlcNAc₂ (FIG. 15). Depending on the cultivationmedium used, the conversion rate of Man₅GlcNAc₂ towardsGlcNAcMan₅GlcNAc₂ also is lower than that observed in the G040 parentstrain. This is most probably related to the slower growth rate observedfor transformants of this Mnn2-Gal10-GalT I fusion protein.

Example 6: Knock-Out of YlALG3 and Simultaneous Overexpression of YlALG6

To allow the generation of a Man₃GlcNAc₂ platform, the ALG3 gene ofstrain G036 (po1d lnuga Δoch1+Hp4d-driven α-1,2-mannosidase) needs to beinactivated. This results into the loss of the ER-localized Alg3pα-1,6-mannosyltransferase activity and changes the composition of thelipid-linked N-glycan precursor structure. Transfer of this structure toan N-glycosylation site of a nascent polypeptide chain makes it possibleto convert the yeast glycosylation profile into mammalian-like N-glycanstructures without the need to express the Mannosidase II. However,since this new lipid-linked structure is not transferred as efficientlyto nascent polypeptides, the Yarrowia ALG6 gene (encoding anER-localized Alg6p α-1,3-glucosyl transferase) needs to be overexpressedsimultaneously to reduce potential protein underglycosylation as much aspossible.

A vector called pYLalg3PUT-ALG6 (FIG. 16) was constructed previously toallow simultaneous knock-out of YlALG3 and Hp4d-driven overexpression ofYlALG6. See U.S. Patent Publication No. 20090069232-A1. A NotI/PacIfragment of this vector, containing this knock-out/knock-in cassette,was transformed into Yarrowia lipolytica G036 and transformants wereselected based on their uracil prototrophy. Clones that had correctlyintegrated the construct were directly screened via N-glycan analysis onthe secretome. Out of 80 screened clones, 2 clones showed anN-glycosylation profile that could fit with the inactivation of YlALG3in a strain expressing an ER-located α-1,2-mannosidase. Apart from afraction Man₃GlcNAc₂ glycans, there was still some Man_(4′)GlcNAc₂ andMan_(5′)GlcNAc₂ as well as a significant amount of glucosylatedN-glycans (GlcMan_(5′)GlcNAc₂ and Glc₂Man_(5′)GlcNAc₂). The latter arethe result of an inefficient trimming by glucosidase II (Grinna andRobbins, J. Biol. Chem. 255, 2255-2258, 1980). The nature of thestructures of Man₄GlcNAc₂ and Man₅GlcNAc₂ was confirmed by in vitrotreatment of the N-glycans with α-1,2-mannosidase (FIG. 17). Dependingon the growth conditions used, the level of Man₃GlcNAc₂ could increaseto up to 60% of the total N-glycan pool, with the glucosylated peaksbeing insensitive towards α-1,2-mannosidase and only slightly sensitivetowards Jack Bean α-mannosidase treatment (aspecific α-mannosidase thatcan act on α-1,2-, α-1,3- and α-1,6-linked mannose residues). Incontrast, the latter enzyme converts the generated Man₃GlcNAc₂ intoMan₁GlcNAc₂ (FIG. 17).

One of the two positive transformants was called G039 and used forfurther glyco-engineering work. The strain was transformed transientlywith pRRQ2 expressing the Cre-recombinase to allow the curing of theURA3 marker that was introduced upon transformation of G036 with vectorpYLalg3PUT-ALG6. Analysis shows that the glycosylation profile remainsthe same after curing. One cured strain was selected for further use anddesignated G045.

Example 7: Expression of GlcNAc-Transferase I in a Man₃GlcNAc₂ ProducingStrain

Similar to what was done in example 3, the introduction of a GnT Iactivity was accomplished via the expression of the Kre2-GnT I fusionprotein. Random integration of such an expression construct for GnT Iwas accomplished in three ways: 1) the non cured strain G039 (seeExample 6) was transformed with the NotI digested vector pYLTmAXhGnTI(Hygr ex) and GnT I expressing clones were initially selected based ontheir ability to survive 300 μg/ml of hygromycin added to the selectionplates, 2) the cured strain G045 (see Example 6) was transformed withthe NotI digested vector pYLTmAXhGnTI (see also Example 3) and GnT Iexpressing clones were initially selected based on their uracilprototrophy or 3) the cured strain G045 (see Example 6) was transformedwith the NotI digested vector pYLHp4mAXhGnTI and GnT I expressing cloneswere initially selected based on their uracil prototrophy. Theconstruction strategy for pYLTmAXhGnTI (Hygr ex) is shown in FIG. 18.When using plasmids pYLTmAXhGnTI (Hygr ex) and pYLTmAXhGnTI, theexpression of GnT I was under the transcriptional control of the TEF1promoter; when using plasmid pYLHp4mAXhGnTI, the GnT I expression wasunder the control of the Hp4d promoter.

Transformation of G039 with pYLTmAXhGnTI (Hygr ex) resulted in threeclones that only emerged on the culture plates after a longer incubationperiod than what was expected. However, analysis of the N-glycosylationprofile of the secretome of these clones showed a clear change in theN-glycan pattern: the Man₃GlcNAc₂ present in the non-transformed G039strain was significantly reduced or almost completely absent while a newpeak, representing an N-glycan with higher molecular weight (about oneglucose unit extra), appeared. Treatment of the isolated N-glycans withJack Bean β-N-acetylhexosaminidase, an enzyme capable of removingterminal β-linked GlcNAc residues, indicated that the new N-glycanindeed is GlcNAcMan₃GlcNAc₂. The new peak disappeared and was completelyconverted into Man₃GlcNAc₂ (FIG. 19). One of the evaluated transformantswas used for further glyco-engineering work and named G047. Similarresults were also obtained when the cured strain G045 was transformedwith pYLTmAXhGnTI (G048) or with pYLHp4mAXhGnTI (G056). Strain G056 wasselected for curing via transient expression of the Cre recombinaseusing plasmid pRRQ2. The resulting strain was called G058.

Depending on the cultivation method used, about 70% of the totalN-glycan pool of strain G047 proved to be GlcNAcMan₃GlcNAc₂ with someremaining Glc₁₋₂Man₅GlcNAc₂ and almost no Man₃GlcNAc₂ was present(conversion rate >>90%) (FIG. 19). Regardless of the high conversionrate, only one copy of the GnT I expression cassette could be identifiedin this strain via Southern blot. Southern analysis was done on BamHIdigested genomic DNA using a DIG-labeled GnT I-specific PCR fragmentthat was generated using primers 5′-GGATGATCACACAATGGCCCTGTTTCTG-3′(SEQID NO:11) and 5′-TGCTCTAGACTAGTTCCAAGAGGGGTC-3′ (SEQ ID NO: 12).

In an alternative strategy, a construct JME925 pPTAde2-URA3ex-Hp4dhGnTIwas generated to allow targeted integration of the Hp4d-driven GnT Iexpression cassette into the ADE2 locus of the Yarrowia genome. Theconstruction strategy is depicted in FIG. 20. Prior to transformation tostrain G045, the plasmid was NotI digested and the targeting/expressioncassette was isolated. Transformants were selected based on theiradenine prototrophy. Correct integration of the expression cassette intothe ADE2 locus was checked via PCR using forward primer Ver1Ade2(5′-CGACGATAGAGCAGGTCTCACTGTTGGGAATGCTG-3′, SEQ ID NO:13) reverse primerVer2Ade2 (5′-CTACACTGACGAAGTGGACATCCCGGCTTGGACTG-3′, SEQ ID NO: 14) andfurther confirmed via Southern blotting. This was done on BamHI/SpeIdigested genomic DNA using a DIG-labeled GnT I-specific PCR fragmentthat was generated using primers 5′-GGATGATCACACAATGGCCCTGTTTCTG-3′ (SEQID NO:15) and 5′-TGCTCTAGACTAGTTCCAAGAGGGGTC-3′ (SEQ ID NO: 16).Synthesis of GlcNAcMan₃GlcNAc₂ onto the secretome was confirmed viaN-glycan analysis and in vitro Jack Bean (β-N-acetylhexosaminidasetreatment (FIG. 21). One GnT I expressing transformant (called G057) wasselected for curing via transient expression of the Cre recombinaseusing plasmid pRRQ2. The resulting strain was called G059.

Example 8: Expression of GlcNAc-Transferase II

A Yarrowia codon-optimized sequence was generated for the expression ofa fusion protein consisting of the first 36 N-terminal amino acids ofthe S. cerevisiae Mnn2 protein (SwissProt AccNo P38069) followed by thecatalytic domain of rat GlcNAc-transferase II (GnT II) (SwissProt AccNoQ09326) (FIG. 22, SEQ ID NO: 17 and SEQ ID NO:18). The yeast Mnn2 36N-terminal amino acids serve as a Golgi localization signal for thecatalytic GnT II domain. In this way, it was ensured that the Mnn2-GnTII fusion protein was localized at the same or even a later position inthe secretion pathway than the Kre2-GnT I (and Mnn2-Man II) fusionprotein and was therefore able to convert GlcNAcMan₃GlcNAc₂ intoGlcNAc₂Man₃GlcNAc₂. The synthetic gene for the expression of the fusionprotein was placed under the transcriptional control of the TEF1promoter, resulting into the plasmids pYLTmAXrGnTII and pYLTmAXrGnTII(ADE2ex). The construction strategy is shown in FIG. 23.

A strain expressing the GnT II activity was generated in two differentways: 1) strain G045 (see Example 6) was transformed simultaneously withNotI digested pYLTmAXhGnTI and NotI digested pYLTmAXrGnTII (ADE2 ex) andtransformants were selected based on their uracil and adenineprototrophy or 2) strain G047 (Example 7) was transformed with NotIdigested pYLTmAXrGnTII (ADE2 ex) and transformants were selected basedon their adenine prototrophy. Integration of the expression cassetteswas checked using forward primer TefPromFW 5′-GTCCCCGAATTACCTTTCC-3′(SEQ ID NO: 19) and reverse primer Lip2TermRV5′-AGGTAGAAGTTGTAAAGAGTG-3′ (SEQ ID NO:20). N-glycan analysis on thesecretome in combination with in vitro treatment of the isolated sugarswith Jack Bean (3-N-acetylhexosaminidase indicated that severaltransformants were capable of producing GlcNAc₂Man₃GlcNAc₂ and thus ofexpressing a functional GnT II activity (FIG. 24). In one selectedcondition, about 40% of the total N-glycan pool consisted ofGlcNAc₂Man₃GlcNAc₂. The conversion rate of the substrateGlcNAcMan₃GlcNAc₂ to GlcNAc₂Man₃GlcNAc₂ was 90%. The final selectedstrains were called G050 (double transformation of G045) and G051 (GnTII expression in G047).

Example 9: Expression of Glucosidase II Alpha and Beta Subunits (Gls2αand Gls213)

Based on the experiments described in Examples 6 to 8, the strategyinvolving the knock-out of YlALG3 and simultaneous overexpression ofYlALG6 results into the generation of N-glycans carrying one or twoterminal glucose residues (Glc₁₋₂Man_(5′)GlcNAc₂). The presence of theseglucose residues hampers the conversion towards Man₃GlcNAc₂ by theER-localized HDEL-tagged α-1,2-mannosidase. In order for the glucoseresidues to be removed, the glucosidase II activity within the ER needsto be increased. In a background without α-1,2-mannosidase expression,overexpression of the Aspergillus niger glucosidase II alpha and betasubunit resulted in the highest conversion of Glc₁₋₂Man₅GlcNAc₂ intoMan_(5′)GlcNAc₂ (U.S. Patent Publication No. 20090069232-A1). Constructsfor the overexpression of the A. niger gls2 subunits were produced asfollows: 1) a Yarrowia codon-optomized cDNA was generated for theexpression of the mature (lacking the signal peptide) A. niger gls2α andgls2β subunit; 2) the cDNA's were cloned in-frame to the Y. lipolyticaLIP2pre-sequence; 3) the resulting LIP2pre-gls2α and LIP2pre-gls2βsequences were cloned under the transcriptional control of theconstitutive TEF1 promoter. The resulting plasmids were calledpYLTUXdL2preAnGlcIIα and pYLeu2ExTEFpreLip2AnGlucIIβ (FIG. 25).

Based on these plasmids, new constructs were generated for thesimultaneous overexpression of the A. niger gls2α and gls2β subunitsunder either TEF1 promoter control (vector JME923pPTura3-LEU2ex-TefL2preAnGlcIIa+b[alt1] for targeted integration—FIG.26) or Hp4d promoter control (vector JME923pPTura3-LEU2ex-Hp4dL2preAnGlcIIa+b[alt1] for targeted integration andvector Zeta-LEU2ex-Hp4dL2preAnGlcIIa+b[alt] for random integration—FIG.27).

Strain G057 (see example 7) was transformed with NotI digested plasmidsJME923 pPTura3-LEU2ex-Hp4dL2preAnGlcIIa+b[alt1] andZeta-LEU2ex-Hp4dL2preAnGlcIIa+b[alt] and transformants were selectedbased on their leucine prototrophy. Several clones were analyzedgenomically via PCR and Southern analysis to evaluate the integration ofthe gls2α and gls2β expression cassette. PCR-analysis and DIG probegeneration for the gls2α subunit was done using primers AnGls2α-FW(5′-GCTGGACTCTTCTTCTATCC-3′) (SEQ ID NO:24) and AnGls2α-RV(5′-GGTCTCCTTCAGAGACAGG-3′) (SEQ ID NO:25); for the gls2β subunit wemade use of primers AnGls2β-FW (5′-CCAAGTTCTACAAGGACACC-3′) (SEQ IDNO:26) and AnGlc2β-RV (5′-CCCTTGACGACCTTAGAGG-3′) (SEQ ID NO:27).Southern analysis to check for targeted integration of the dualHp4dGls2α/β expression cassette was done on Eco47III-digested gDNA whenusing the gls2α probe, and on SpeI/SfiI-digested gDNA when using thegls2β probe. The majority of the selected clones showed correctintegration of the dual expression cassette into the URA3 locus.Southern analysis for random integration of the dual Hp4dGls2α/βexpression cassettes was checked on PvuI-digested gDNA with both probes.In all cases, only one copy of the dual expression cassette wasintegrated.

Next, N-glycan analysis was performed on several clones confirmed tohave the dual Hp4dGls2α/β expression cassette (correctly) integrated.N-glycosylation was examined on total secreted protein after three daysof falcon cultivation. Several clones showed a significant reduction ofthe glucosylated sugars and an increase of Man₃GlcNAc₂ andGlcNAcMan₃GlcNAc₂. The profiles of a clone that has integrated the dualexpression cassette randomly (=strain G060) on the one hand and in atargeted way (=strain G061) on the other, are shown in FIG. 28. The twosmaller peaks represent Man_(4′)GlcNAc₂ and Man_(5′)GlcNAc₂, since theyshift to Man₃GlcNAc₂ resp. Man₁GlcNAc₂ upon treatment withα-1,2-mannosidase and Jack Bean mannosidase. The latter treatment alsoresults in a partial conversion of the remaining Glc₁₋₂Man_(5′)GlcNAc₂into Glc₁₋₂Man_(4′)GlcNAc₂ and of GlcNAcMan₃GlcNAc₂ intoGlcNAcMan₂GlcNAc₂. Presence of Man_(4′)GlcNAc₂ and Man_(5′)GlcNAc₂however indicates incomplete conversion towards Man₃GlcNAc₂ by theheterologously co-expressed HDEL-tagged α-1,2-mannosidase. Similarly,the presence of Man₃GlcNAc₂ indicates incomplete transfer of aGlcNAc-residue by recombinant human GnT I to obtain GlcNAcMan₃GlcNAc₂.However, based on results described above (e.g. G047 cultivation inExample 7, FIG. 19), it is clear that differences in cultivationconditions can increase the conversion rates significantly and thusimprove the end result.

Example 10: Expression of GlcNAc-Transferase II in the GlcNAcMan₃GlcNAc₂Producing Strain G061

As described in Example 8, a Yarrowia codon-optimized sequence wasgenerated for the expression of a fusion protein consisting of the first36 N-terminal amino acids of the S. cerevisiae Mnn2 protein (SwissProtAccNo P38069) followed by the catalytic domain of rat GlcNAc-transferaseII (GnT II) (SwissProt AccNo Q09326) (FIG. 22, SEQ ID NO:17 and SEQ IDNO:18, respectively). The yeast Mnn2 36 N-terminal amino acids serve asa Golgi localization signal for the catalytic GnT II domain. In thisway, it was ensured that the Mnn2-GnT II fusion protein was localized atthe same or even a later position in the secretion pathway than theKre2-GnT I fusion protein and was therefore able to convertGlcNAcMan₃GlcNAc₂ into GlcNAc₂Man₃GlcNAc₂. The synthetic gene for theexpression of the fusion protein was placed under the transcriptionalcontrol of the Hp4d promoter resulting in plasmid pYLHp4mAXrGnTII, whichwas used for random integration of the Hp4d-driven GnT II expressioncassette into the Yarrowia genome. In an alternative strategy, constructOXYP289 pPTAxp1-ADE2ex-Hp4dhGnTII was generated to allow targetedintegration of the Hp4d-driven GnT II expression cassette into the AXP1locus of the Yarrowia genome.

Prior to transformation of strain G061 (see Example 9), the plasmidswere NotI digested and the targeting/expression cassette was isolated.Transformants were selected based on their adenine prototrophy. Correctintegration of the expression cassette into the ADE2 locus was confirmedby Southern blot analysis after digesting the genomic DNA with XmnI. ADIG-labeled probe with specificity for the GnT II coding sequence wasgenerated using forward primer rGnTII-FW (5′-GACCAGATGCTGCGAAACG-3′)(SEQ ID NO: 28) and reverse primer rGnTII-RV (5′-CTTGACGTCCACCTTGTCG-3′)(SEQ ID NO: 29). This strategy produces a band of 3172 bp when the geneis successfully integrated into the Axp1 locus.

In an alternative strategy, correct integration into the Axp1 locus canbe examined via a PCR reaction on genomic DNA using the forward primerAXPVer1b (5′-GCCTGAACGGCACGATGCGATCGTGGCAATCC-3′) (SEQ ID NO: 30) andthe reversed primer AXPVer2b (5′-CAAGAAGCCTCAGGCTCGGCGAATCTCCA TC-3′)(SEQ ID NO: 31). In case of correct targeting into the Axp1 locus, a PCRfragment of 6489 bp is expected.

N-glycan analysis on the secretome, in combination with in vitrotreatment of the isolated sugars with Jack Bean(β-N-acetylhexosaminidase or T. reesei α-1,2-mannosidase, indicated thatseveral transformants were capable of producing GlcNAc₂Man₃GlcNAc₂ andthus of expressing a functional GnT II activity (FIG. 29). The analysesindicated that about 25 to 30% of the total N-glycan pool consisted ofGlcNAc₂Man₃GlcNAc₂, with a GlcNAcMan₃GlcNAc₂ to GlcNAc₂Man₃GlcNAc₂conversion rate of about 90%. The final selected strains were calledG070 (integration of pYLHp4mAXrGnTII into G061) and G071 (integration ofOXYP289 pPTAxp1-ADE2ex-Hp4dhGnTII into G061).

Example 11: Construction of a Tandem Plasmid for SimultaneousHp4d-Driven Expression of the Anti-HER2 Heavy Chain (HC) and Light Chain(LC) into Yarrowia lipolytica

The amino acid sequences for the anti-HER2 antibody heavy and lightchains were obtained from Carter et al., Proc Natl Acad Sci USA.,89(10): 4285-4289 (1992); and Ward et al., Appl Environ Microbiol.,70(5): 2567-2576 (2004). The relevant amino acid sequences were reversetranslated, codon-optimized for Yarrowia lipolitica, and synthesized byGenArt, Regensburg Germany. Regions of very high (>80%) or very low(<30%) GC content were avoided where possible. During the optimizationprocesses, the following cis-acting sequence motifs were avoided:internal TATA-boxes, chi-sites and ribosomal entry sites, AT-rich orGC-rich sequence stretches, repeat sequences and RNA secondarystructures as well as (cryptic) splice donor and acceptor sites. Inorder to allow secretion of the ectopic proteins, the coding sequence ofthe Lip2 protein ‘prepro’ signal (followed by that of a peptide linker‘GGG’) was added to the 5′ region of the coding sequences. ‘GGG’ wasadded to enhance the changes for correct Kex2 processing. FIG. 30Acontains the nucleotide sequence of the synthetic preproLip2-LC(=750 bp)(SEQ ID NO: 32). FIG. 30B contains the amino acid sequence of thepreproLip2-LC(=250 Aa; MW=27.011 Da; pI=8.46) (SEQ ID NO: 33). FIG. 31Acontains the nucleotide sequence of the synthetic preproLip2-HC(=1458bp) (SEQ ID NO: 34). FIG. 31B contains the amino acid sequence of thepreproLip2-HC(=486 Aa; MW=52.853 Da; pI=8.65) (SEQ ID NO: 35). Thecoding sequences for preproLip2-HC and -LC were introduced into the samevector, called pYLHp4L2preproHerHC/LC (GUT2ex)-ori2.

Example 12: Expression of the Anti-HER2 Antibody HC and LC into Yarrowialipolytica Strains with a Varying Degree of Glyco-Engineering

Plasmid pYLHp4L2preproHerHC&LC (GUT2ex)-ori2 was digested with NotI andthe HC-/LC-tandem expression cassette was isolated before transformingYarrowia lipolytica strains G045, G057, G061 and G071 (see Table 2).Transformants containing the randomly integrated HC-/LC-expressioncassette were selected based on their ability to grow on glycerol as thesole carbon source. Expression analysis of the HC and LC was done viawestern blotting after a 4 day shake flask cultivation of the selectedtransformants in rich medium containing glycerol as the only carbonsource (SuperT/glycerol medium: 0.5% yeast extract; 2% malt extract; 1%trypton; 1.5% glycerol; 200 mM phosphate buffer pH 6.8). LC-detectionwas performed using a mouse monoclonal to Kappa Free Light Chains (4C11)(Abcam) while HC-detection was done using mouse monoclonal anti-humanIgG (γ-chain specific) (Sigma).

The N-glycans of the secretome of the anti-HER2 antibody producingstrains showed a similar profile as the corresponding glyco-engineeredstrains that were not expressing any HC and LC (FIG. 32). Thepercentages of N-glycans in strains with the G045, G057, G061, and G071background were determined after a 6-day shake flask cultivation inSuperT/glycerol medium. In a G045 background, 54.6% of the N-glycanswere Man₃GlcNAc₂. In the G057 background, 47.5% of the N-glycans wereGlcNAc₁Man₃GlcNAc₂. In a G061 background, 58.9% of the N-glycans wereGlcNAc₁Man₃GlcNAc₂. In a G071 background, 37.6% of the N-glycans wereGlcNAc₂Man₃GlcNAc₂.

Example 13: Fermentation of Yarrowia Strain G096, a GlcNAc₂Man₃GlcNAc₂Synthesizing Strain Expressing the Anti-HER2 Antibody HC and LC

Several pYLHp4L2preproHerHC&LC (GUT2ex)-ori2 transformants of Yarrowialipolytica G071, a strain capable of synthesizing GlcNAc₂Man₃GlcNAc₂,were analyzed for HC and LC expression levels. One of these clones,G096, was chosen for further analysis.

Fermentation was done in a 14-litre stirred tank bioreactor (MAVAG AG)equipped with a process control and management system (Lucillus PIMS).The relative partial oxygen pressure in the medium, the CO₂ and O₂concentrations in the exhaust gas, pH value, temperature, reactoroverpressure, reactor weight, feed weight and base weight were allmonitored on-line. Foam generation was counteracted by adding theantifoaming agent polypropylene glycol (PPG). Adjustments in pH weredone by either the addition of a 25% ammonia solution or a 8.5%phosphoric acid solution.

A seed culture of G096 was grown at 28° C. in a shake flask containingrich medium. The seed culture was inoculated into the fermentorcontaining mineral medium to start a batch phase at 28° C. withunrestricted growth, using glycerol as only carbon source. This phasewas used to rapidly reach a high biomass concentration. From that pointonward, the process was shifted to an exponential glycerol fed batch(with glycerol as sole carbon and energy source; pH 6), with a constantgrowth rate of 0.02. As an example, the results for a fed batchfermentation at 28° C. are described below.

The fed-batch phase lasted for 148 hours. At different time-points ofthe fermentation, samples were taken to follow up the followingparameters: 1) expression of the LC and HC protein backbones via westernblot; 2) expression of functional anti-HER2 antibody via an ELISA; and3) evolution of the N-glycosylation profile of the secretome. Thefull-length HC expression level reached a maximum around timepoint 7 (39hrs) and remains approximately equal from then onwards. The LCexpression reached a maximum between time-points 7 (39 hrs) and 10 (73hrs), but decreased somewhat in the later time-points. Some LC-dimerswere produced between time-points 5 (25 hrs) and 9 (62 hrs), butdisappeared again from that point onwards.

A functional ELISA was developed to measure the production of anti-HER2antibody that has at least one functional antigen binding domain. Plateswere coated with a recombinant variant of the natural HER2 antigen, therecombinant human ErbB2/Fc chimera (R&D systems). Then a dilution of themedium, harvested at different time-points, was added to the coatedplates. Assessment of the amount of antigen binding protein was doneusing a HRP-conjugated anti-human kappa LC antibody (Sigma). Theevolution of the amount of ErbB2/Fc chimera binding protein (a measureof the amount of secreted functional anti-HER2 antibody) within thefed-batch fermentation is shown in FIG. 33. The data show a gradualincrease in the levels of anti-HER2 antibody, with a maximum of 10 to 12mg/L at the end of the production phase.

N-glycan analysis was done on samples taken at several time-pointsduring the fed-batch fermentation. The results are shown in FIGS. 34Aand 34B. At the beginning of the fed-batch phase, there was asignificant amount of glucose-containing N-glycans present. Fromtime-point 6 onward (34 hrs after start of exponential feeding), thelevel of glucosylated N-glycans decreased significantly with hardly anyleft at the time of harvest (time-point 18, 148 hrs). This indicatedthat proteins originally carrying glucose-containing N-glycans, werediluted out by the end of the fermentation. At that point about 86% ofthe N-glycans isolated from the secretome had the structureGlcNAc₂Man₃GlcNAc₂.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. An isolated Yarrowia lipolytica cell geneticallyengineered to produce proteins comprising GlcNAc2Man3GlcNAc2 N-glycans,wherein said Yarrowia lipolytica cell is genetically engineered tocomprise the following alterations: a) deficient in Outer Chainelongation (OCH1) activity; b) deficient in Asparagine linkedGlycosylation 3 (ALG3) activity; c) recombinantly expresses anα-1,2-mannosidase inclusive of a targeting sequence to an intracellularcompartment; d) recombinantly expresses Asparagine Linked Glycosylation6 (ALG6); and e) recombinantly expresses a GlcNAc-transferase I andGlcNAc-transferase II, wherein both transferases are inclusive of atargeting sequence to an intracellular compartment, wherein as a resultof said alterations, said Yarrowia lipolytica cell produces proteinscomprising GlcNAc₂Man₃GlcNAc₂ N-glycans.
 2. The Yarrowia lipolytica cellof claim 1, wherein said genetically engineered Yarrowia lipolytica cellfurther comprises a nucleic acid encoding a target protein, wherein saidcell produces said target protein modified to comprise saidGlcNAc₂Man₃GlcNAc₂N-glycans.
 3. The Yarrowia lipolytica cell of claim 1,said Yarrowia lipolytica cell further comprising a nucleic acid encodingthe α and β subunits of a Glucosidase II, wherein expression of said αand β subunits of said Glucosidase II in said Yarrowia lipolytica cellproduces said protein comprising said GlcNAc₂Man₃GlcNAc₂ N-glycans. 4.The Yarrowia lipolytica cell of claim 1, wherein said GlcNAc-transferaseI and GlcNAc-transferase II are targeted to the Golgi apparatus.
 5. TheYarrowia lipolytica cell of claim 1, wherein said α-1,2-mannosidase istargeted to the endoplasmic reticulum.
 6. A pure culture comprisingYarrowia lipolytica cells, according to claim
 1. 7. The culture of claim6, said Yarrowia lipolytica cell further comprising a nucleic acidencoding the α and β subunits of a Glucosidase II, wherein expression ofsaid α and β subunits of said Glucosidase II in said Yarrowia lipolyticacell produces said protein comprising said GlcNAc₂Man₃GlcNAc₂ N-glycans.8. The culture of claim 6, wherein said GlcNAc-transferase I andGlcNAc-transferase II are targeted to the Golgi apparatus.
 9. Theculture of claim 6, wherein said α-1,2-mannosidase is targeted to theendoplasmic reticulum.